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Research Article| Volume 298, ISSUE 5, 101919, May 2022
CPR-C4 is a highly conserved novel protease from the Candidate Phyla Radiation with remote structural homology to human vasohibins
Katy A.S. Cornish
Department of Chemistry, Durham University, Lower Mountjoy, Durham, County Durham, United Kingdom
Joanna Lange
Bio-Prodict, Nijmegen, The Netherlands
Arnthór Aevarsson
Matis ohf, Reykjavik, Iceland
Ehmke Pohl
For correspondence: Ehmke Pohl
Department of Biosciences, Durham University, Upper Mountjoy, Durham, County Durham, United Kingdom
Open AccessPublished:April 08, 2022DOI:https://doi.org/10.1016/j.jbc.2022.101919
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The Candidate Phyla Radiation is a recently uncovered and vast expansion of the bacterial domain of life, made up of largely uncharacterized phyla that lack isolated representatives. This unexplored territory of genetic diversity presents an abundance of novel proteins with potential applications in the life-science sectors. Here, we present the structural and functional elucidation of CPR-C4, a hypothetical protein from the genome of a thermophilic Candidate Phyla Radiation organism, identified through metagenomic sequencing. Our analyses revealed that CPR-C4 is a member of a family of highly conserved proteins within the Candidate Phyla Radiation. The function of CPR-C4 as a cysteine protease was predicted through remote structural similarity to the Homo sapiens vasohibins and subsequently confirmed experimentally with fluorescence-based activity assays. Furthermore, detailed structural and sequence alignment analysis enabled identification of a noncanonical cysteine-histidine-leucine(carbonyl) catalytic triad. The unexpected structural and functional similarities between CPR-C4 and the human vasohibins suggest an evolutionary relationship undetectable at the sequence level alone.
thermophile
cysteine protease
protein crystallization
structure-function
protein evolution
Candidate Phyla Radiation
CPR (Candidate Phyla Radiation), TSA (Thermal shift analysis)
The Candidate Phyla Radiation (CPR) is a vast collection of phyla and superphyla in the bacterial domain of life that currently lack isolated representatives (
Castelle C.J.
Banfield J.F.
Major new microbial groups expand diversity and alter our understanding of the tree of life.
Kantor R.S.
Wrighton K.C.
Handley K.M.
Sharon I.
Hug L.A.
Thomas B.C.
Small genomes and sparse metabolisms of sediment-associated bacteria from four candidate phyla.
mBio. 2013; 4e00708-13
). There is on-going debate over the expanse of this radiation, with some predicting that the CPR comprises half of all bacterial diversity within the tree of life (
Baker B.J.
Anantharaman K.
Brown C.T.
Probst A.J.
Butterfield C.N.
Hernsdorf A.W.
Amano Y.
Ise K.
Suzuki Y.
Dudek N.
Relman D.A.
Finstad K.M.
Amundson R.
A new view of the tree of life.
Nat. Microbiol. 2016; 1: 16048
Parks D.H.
Rinke C.
Chuvochina M.
Chaumeil P.-A.
Woodcroft B.J.
Evans P.N.
Hugenholtz P.
Tyson G.W.
Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life.
Nat. Microbiol. 2017; 2: 1533-1542
Schulz F.
Eloe-Fadrosh E.A.
Bowers R.M.
Jarett J.
Nielsen T.
Ivanova N.N.
Kyrpides N.C.
Woyke T.
Towards a balanced view of the bacterial tree of life.
Microbiome. 2017; 5: 140
). As such, these candidate phyla have earned the nickname 'microbial dark matter', likely accounting for a significant portion of the Earth's biomass but with largely unknown properties at present (
Schwientek P.
Sczyrba A.
Anderson I.J.
Cheng J.-F.
Darling A.
Malfatti S.
Swan B.K.
Gies E.A.
Dodsworth J.A.
Hedlund B.P.
Tsiamis G.
Sievert S.M.
Liu W.-T.
Insights into the phylogeny and coding potential of microbial dark matter.
Marcy Y.
Ouverney C.
Bik E.M.
Losekann T.
Ivanova N.
Martin H.G.
Szeto E.
Platt D.
Quake S.R.
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While genome sizes and metabolic capabilities within a phylum are usually highly variant, organisms within the CPR have consistently small genomes (from 0.7 – 1.2 Mbp) and share a similarly limited set of biosynthetic pathways (
Miller C.S.
VerBerkmoes N.C.
Wilkins M.J.
Hettich R.L.
Lipton M.S.
Williams K.H.
Long P.E.
Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla.
Luef B.
Frischkorn K.R.
Holman H.N.
Birarda G.
Singh A.
Siegerist C.E.
Tringe S.G.
Downing K.H.
Comolli L.R.
Diverse uncultivated ultra-small bacterial cells in groundwater.
). It is likely that the limited metabolisms of the CPR and possibly a strict requirement for symbiotic lifestyles, are responsible for the difficulties associated with their cultivation. In addition, the ribosomal RNA sequences of CPR organisms have been reported to contain highly uncommon features, including encoded proteins and self-splicing introns (
Unusual biology across a group comprising more than 15% of domain Bacteria.
). There is also substantial divergence in 16S ribosomal RNA sequences in CPR species. As these sequences are the key for uncovering new species by PCR techniques, it is estimated that at least half of the organisms sampled from the CPR would remain undetected in typical PCR surveys (
). These unusual properties point to atypical biology for a major part of the bacterial domain and have played a major part in the CPR remaining undetected for so long.
As well as improving our understanding of this newly uncovered yet expansive region of the tree of life, the exploration of novel gene products is the key for discovering enzymes that present new or unusual functions, as exemplified by the discovery of the CRISPR-Cas system (
Makarova K.S.
Haft D.H.
Barrangou R.
Brouns S.J.J.
Charpentier E.
Horvath P.
Moineau S.
Mojica F.J.M.
Wolf Y.I.
Yakunin A.F.
van der Oost J.
Koonin E.V.
Evolution and classification of the CRISPR–Cas systems.
Nat. Rev. Microbiol. 2011; 9: 467-477
). The hunt for genomes in extreme natural environments by the Virus-X consortium has unearthed and categorized over 50 million genes using a metagenomic approach (
Aevarsson A.
Kaczorowska A.-K.
Adalsteinsson B.T.
Ahlqvist J.
Al-Karadaghi S.
Altenbuchner J.
Arsin H.
Átlasson Ú.Á.
Brandt D.
Cichowicz-Cieślak M.
Cornish K.A.S.
Courtin J.
Dabrowski S.
Dahle H.
Djeffane S.
Going to extremes – a metagenomic journey into the dark matter of life.
FEMS Microbiol. Lett. 2021; 368fnab067
). This method involves sequencing all of the genetic information isolated from a natural environment, including that which has previously evaded detection (
). The use of metagenomics techniques exploiting next generation sequencing technologies eliminates the need for cultivation and has made the genetic content of the CPR much more accessible for exploration (
Rappé M.S.
Giovannoni S.J.
The uncultured microbial majority.
Annu. Rev. Microbiol. 2003; 57: 369-394
He C.
Whittaker M.L.
Farag I.F.
Doudna J.A.
Cate J.H.D.
Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems.
Nat. Microbiol. 2021; 6: 354-365
). In the Virus-X project, the natural systems explored include Icelandic geothermal hot springs and the Loki's Castle hydrothermal vent system on the Mid-Atlantic ridge between Greenland and Norway. The bioprospecting activities concentrated on genomes from such extreme habitats as sources of novel enzymes with desirable properties, including tolerance to extremes of temperature, pH, and salt concentration, and high innovation potential for applications in the life sciences industry, such as biotechnology and pharmaceuticals. Particular focus was given to hypothetical or 'unknown' gene products with undetermined properties and potentially new functionalities. The functions of these hypothetical proteins are impossible to predict from nucleotide sequence alone due to sequence divergence and a lack of detectable homology. As such, structure determination offers a clearer insight into potential function and understanding of protein families within the CPR. Structure-based multiple sequence alignments consequently enable direct comparisons that can uncover structural similarities between proteins in spite of low sequence identity.
This study focusses on one predicted gene product from the genome of the first identified thermophilic CPR bacterium, discovered in an Icelandic hot spring. This gene product was denoted CPR-C4 and annotated as a hypothetical protein, with no characterized biological function. The lack of sequence identity to any proteins of known function in public databases, along with the identification of hundreds of related hypothetical protein sequences, led to the categorization of CPR-C4 as a high-priority target for structural determination with a potentially novel function or mechanism. In this article, we present the X-ray crystal structure of CPR-C4 solved by multiple-wavelength anomalous diffraction techniques at 2.60 Å and determined in three crystal forms to a maximum resolution of 2.25 Å. Subsequent deep sequence and structural analyses revealed an unexpected similarity to the Homo sapiens tubulin-tyrosine carboxypeptidases despite no significant sequence identity. This enabled the functional annotation of CPR-C4 as a protease with an unusual cysteine-histidine-leucine(carbonyl) catalytic triad and points toward an intriguing evolutionary relationship in this cysteine protease superfamily.
Target identification and sequence analysis
The CPR-C4 gene was identified through metagenomic sequencing of a water sample from a terrestrial hot-spring in Iceland, with conditions of 75 °C and pH 6.0. The gene is one of 442,373 sequenced genes in the total metagenome from this sample, forming part of a contig of over 400 kbp in length and comprised of 432 genes. This contig was classified as belonging to a bacterial organism within the CPR through taxonomic annotation (Fig. S1). BLAST (
Altschul S.F.
Gish W.
Miller W.
Myers E.W.
Lipman D.J.
BLAST: Basic local alignment search tool.
) searches enabled assignment of known or predicted functions to only 30% of the contig gene products. CPR-C4 was part of the remaining 70% for which it was not possible to identify a putative function through sequence alone, resulting in its annotation as a hypothetical protein. When classifying the genes based on the phylum classification level of their most significant BLAST (
) hit, we found that almost three quarters were categorized as either Candidatus Adlerbacteria or some unclassified bacterial phylum, with the remainder mostly from other 'Candidatus' phyla.
The CPR-C4 gene product was of particular interest as a hypothetical protein, with unknown function but showing extended conservation across CPR and non-CPR bacteria. Although searches of publicly available databases did not lead to assignment of any enzymatic domains or uncover potential structural homologs, BLAST (
) identified hundreds of hypothetical proteins with significant sequence similarity to CPR-C4 (over 400 with an E value < 1e−50). The CPR-C4 gene and its neighbors are shown in Figure 1A, which indicates that CPR-C4 is transcribed in the opposite direction to its flanking genes. Genes encoding proteins for ABC transport systems were identified adjacent to CPR-C4, though similar genes were not identified in proximity to CPR-C4 homologs in Candidatus Kaiserbacteria and Candidatus Adlerbacteria genomes. The gene position therefore does not offer any potential function for CPR-C4, though the sequence analysis points to an important and conserved protein in the CPR and beyond.
Figure 1CPR-C4 gene location and residue conservation. A, region of the contig containing the CPR-C4 gene and its neighbors, with predicted functions of gene products stated where annotation has been possible. Predicted genes are depicted as arrows following the strand direction of transcription and are color coded by the phylum classification level of their most significant BLAST (
) hit: yellow = unclassified bacteria; pale purple = Candidatus Adlerbacteria; white = unknown. B, amino acid sequence of the CPR-C4 protein, with residues conserved in ≥90% of instances in the CP01A subfamily shown in blue. Residues falling within the subfamily core region are underlined. 99.9% conserved residues C61 and H93 (following CPR-C4 numbering) are boxed in red. C, percentage conservation of residue identities within the CP01A subfamily core region generated using the 3DM alignment statistics tool following 3DM numbering. Green bars represent conservation of over 50% at that residue position; gray bars show conservation less than 50% at that position. The 99.9% conserved C61 and H93 (CPR-C4 numbering) are at positions 62 and 94, respectively. CPR, Candidate Phyla Radiation.
Deeper sequence analysis with the 3DM software (
Kuipers R.K.
Joosten H.-J.
van Berkel W.J.H.
Leferink N.G.H.
Rooijen E.
Ittmann E.
van Zimmeren F.
Jochens H.
Bornscheuer U.
Vriend G.
dos Santos V.A.P.M.
Schaap P.J.
3DM: Systematic analysis of heterogeneous superfamily data to discover protein functionalities.
Proteins. 2010; 78: 2101-2113
) generated a subfamily for CPR-C4, denoted CP01A, containing 794 protein sequences. Twenty five of these, including CPR-C4, were identified by the Virus-X consortium. Of the 221 residues in the CPR-C4 sequence, the first 215 were found to fall within a conserved 'core region' in the subfamily (Fig. 1B). The extent of conservation in this core region is shown in Figure 1C. Relative to a highly conserved center, residues at the N and C termini are increasingly variable across the CP01A subfamily. The overall conservation of sequence in the subfamily is stark, with over half (110/216) of the residue identities conserved in ≥50% of the 794 subfamily sequences and 15% (32/216) of residues conserved in ≥90% of instances. This analysis highlights that CPR-C4 is part of a family of proteins with significant sequence conservation but unknown function; however, several residues can be identified that are almost entirely conserved and, as such, can be considered important for function. For example, C61 and H93 (CPR-C4 sequence numbering, Fig. 1B), residue types often involved in enzymatic activities, are 99.9% conserved in the subfamily. Structure determination was necessary to uncover the overall fold, analyze residue interactions, and assess the influence of these highly conserved residues, to uncover protein function.
Protein production and biophysical characterization
Preliminary small-scale experiments confirmed that the 'hypothetical' CPR-C4 protein could be induced to overexpress in an E. coli host, though initial expression and purification experiments resulted in low protein yields, producing less than 0.5 mg of soluble protein per l of culture. It was also noticeable that CPR-C4 bound tightly to the immobilized Ni2+ column during purification, requiring an unusually high concentration of imidazole (>500 mM) to elute. SDS-PAGE analysis and electrospray ionization mass spectrometry confirmed the identity of CPR-C4 based on expected molecular weight (27,121 Da, Fig. S2), and size-exclusion chromatography identified that the protein exists in dimeric form (Fig. S3).
Thermal shift analysis
Thermostability is a highly desirable feature of proteins for biotechnology applications (
Zamost B.L.
Nielsen H.K.
Starnes R.L.
Thermostable enzymes for industrial applications.
J. Ind. Microbiol. 1991; 8: 71-81
Rigoldi F.
Donini S.
Redaelli A.
Parisini E.
Gautieri A.
Review: Engineering of thermostable enzymes for industrial applications.
APL Bioeng. 2018; 2: 011501
Elleuche S.
Schrö C.
Sahm K.
Antranikian G.
Extremozymes-biocatalysts with unique properties from extremophilic microorganisms.
). Thermal shift analysis (TSA) can be used to assess the intrinsic thermotolerance of proteins, as well as identify additives and buffer systems that can improve thermostability (
Grøftehauge M.K.
Hajizadeh N.R.
Swann M.J.
Pohl E.
Protein-ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI).
Acta Crystallogr. D. Biol. Crystallogr. 2015; 71: 36-44
Bruce D.
Cardew E.
Freitag-Pohl S.
How to stabilize protein: Stability screens for thermal shift assays and nano differential scanning fluorimetry in the virus-X project.
J. Vis. Exp. 2019; https://doi.org/10.3791/58666
). TSA was conducted to establish stabilizing conditions for CPR-C4 in order to improve yields and prevent precipitation, which made room temperature purification challenging. Purification with a phosphate-based buffer system, in place of the Hepes buffer used in initial experiments, increased the amount of soluble protein obtained after purification, reflected by a stabilization of approximately 4 °C in phosphate relative to Hepes in TSA with the Durham pH Screen.
The TSA results of CPR-C4 with the Durham Salt Screen are represented in Figure 2. The majority of additives screened resulted in some level of stabilization relative to the reference melt temperature, Tm, in water. The protein is markedly stabilized by a range of inorganic salts, including several sodium and ammonium salts that caused a ≥20 °C increase in Tm at the highest concentration tested (Fig. 2A). This consistent increase in thermotolerance shows that CPR-C4 is stabilized by high salt concentrations, with negligible preference for chemical composition.
Figure 2Thermal shift analysis of CPR-C4. A, results from TSA of CPR-C4 with the Durham Salt Screen showing changes in melt temperature, Tm, upon addition of stabilizing salts at increasing concentrations (n = 3, ± st. dev.). B, normalized fluorescence intensities at 590 nm for TSA of CPR-C4 in water (blue) and with addition of 1 mM ZnCl2 (red). Raw experimental data are shown as individual points; dashed vertical lines indicate the calculated Tm values of CPR-C4 in water (blue) and with addition of 1 mM ZnCl2 (red) (
). CPR, Candidate Phyla Radiation; TSA, Thermal shift analysis.
While CPR-C4 is not intrinsically thermostable, with an unexpectedly low reference Tm of only 29.1 ± 0.5 °C (n = 6), it was hugely stabilized by the addition of divalent metal cations. This is most dramatic with the addition of ZnCl2, as demonstrated by an increase in Tm of over 30 °C (Tm 60.9 ± 0.2 °C, n = 3, Fig. 2B). Consequently, protein yields were improved more than 20-fold with the addition of 100 μM ZnCl2 to expression media, producing >10 mg of purified protein per l of culture. The increase in soluble protein yield resulting from these small alterations to expression and purification procedures after TSA was critical for downstream assays and demonstrates the stabilizing effect of Zn2+ on CPR-C4.
The CPR-C4 protein, including an N-terminal His6-tag (Fig. S4), crystallized in two space groups with different crystal morphologies but similar resolution limits (Table 1). X-ray fluorescence scans identified an unambiguous Zn signal from crystal form 1, and the subsequent X-ray absorption edge scan (Fig. S5) was used to select appropriate wavelengths for data collection, optimizing the collection of an anomalous Zn signal for multiple wavelength anomalous diffraction phasing (
Cha S.S.
An Y.J.
Jeong C.S.
Kim M.K.
Lee S.G.
Lee K.H.
Oh B.H.
Experimental phasing using zinc anomalous scattering.
Acta Crystallogr. Sect. D Biol. Crystallogr. 2012; 68: 1253-1258
). It should be noted that these crystals were grown using protein expressed without the addition of Zn to media. The crystals diffracted to a maximum resolution of 2.60 Å in P3221, with two protein chains generating the functional dimer showing two-fold rotational noncrystallographic symmetry and two Zn2+ ions in the asymmetric unit. The structure solution was nontrivial: model building after Zn substructure solution with SHELXD (
Sheldrick G.M.
A short history of SHELX.
Acta Crystallogr. Sect. A. Found. Crystallogr. 2008; 64: 112-122
) required data processed with autoPROC using STARANISO (
Vonrhein C.
Tickle I.J.
Flensburg C.
Keller P.
Paciorek W.
Sharff A.
Bricogne G.
Advances in automated data analysis and processing within autoPROC , combined with improved characterisation, mitigation and visualisation of the anisotropy of diffraction limits using STARANISO.
Acta Crystallogr. Sect. A. Found. Adv. 2018; 74: a360
Smart O.
Womack T.
Data processing and analysis with the autoPROC toolbox.
Acta Crystallogr. Sect. D Biol. Crystallogr. 2011; 67: 293-302
). The density for chain A, which was built first, was notably better defined so that even the side chains of the His6-tag were interpretable. Crystal form 2 diffracted to a resolution of 2.68 Å in space group I222, with one polypeptide chain per asymmetric unit, and was solved by molecular replacement with the model from crystal form 1. To assess the effect of the non-native His6-tag, crystallization was also subsequently conducted using protein without the tag (Fig. S4D), resulting in a third crystal form in space group P212121 and improved resolution (2.25 Å), with one dimer per asymmetric unit. Interface and assembly analysis of the form 1 CPR-C4 dimer with PISA (
) showed an interface area of 1669.1 Å2 (15%) and a binding energy of −26.2 kcal mol−1. This strongly suggests that this interface is part of the biological assembly of the protein and, alongside size exclusion analysis (Fig. S3), confirms that the protein exists naturally in dimeric form.
Table 1Crystallographic data collection and refinement statistics
Beamline DLS I03 DLS I24 DLS I04
Wavelength (Å) 1.2824 1.2819 0.979
Resolution range (Å) 48.26–2.60 (2.71–2.60) 67.40–2.68 (2.81–2.68) 44.26–2.25 (2.32–2.25)
Space group P3221 I222 P212121
Unit cell dimensions (Å), (°) 123.39, 123.39, 96.53, 90, 90, 120 56.31, 79.73, 126.20, 90, 90, 90 44.78, 77.55, 161.72, 90, 90, 90
No. of reflections 224,888 (26,437) 79,870 (8047) 337,006 (16,676)
Unique reflections 26,494 (3143) 8223 (995) 27,119 (2058)
Multiplicity 8.5 (8.4) 9.7 (8.1) 12.4 (8.1)
Completeness (%) 99.8 (99.2) 98.8 (92.7) 98.1 (85.3)
⟨I/σ(I)⟩ 14.4 (0.7) 7.4 (1.4) 17.1 (1.1)
Wilson B-factor (Å2) 75.3 45.3 55.9
Rmerge 0.094 (4.086) 0.453 (6.072) 0.085 (1.770)
Rpim 0.050 (2.198) 0.153 (2.225) 0.025 (0.666)
CC1/2 1.000 (0.368) 0.972 (0.055) 0.999 (0.360)
Rwork 0.234 0.238 0.179
Rfree 0.285 0.259 0.225
Number of atoms 3412 1671 7223
Number of protein residues 435 211 433
RMSD bond lengths (Å) 0.0060 0.0061 0.0076
RMSD bond angles (°) 1.418 1.406 1.442
Ramachandran plot favored/allowed/outliers 409/18/0 197/9/1 407/19/0
Average B factor, overall (Å2) 109.0 51.0 61.0
PDB accession codes 7OB6 7OB7 7PJO
Values in parentheses are for the highest resolution shell.
In all crystal forms, the structure of CPR-C4 is a homodimer displaying a mixed αβ fold, with a central curved antiparallel β-sheet surrounded by short α-helices connected by flexible loops (Fig. 3A). Surprisingly, the two Zn2+ ions were only present in crystal form 1 and were both found to interact with the same chain (chain A, Fig. 3B), in a tetrahedral geometry, as is most common for Zn2+ coordination (
Harding M.M.
Geometry of metal-ligand interactions in proteins.
Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 401-411
). The first is coordinated by three aspartate side chains (D83, D92, D182) and a water molecule (Fig. 3C), and the second is coordinated by two histidine residues from the N-terminal His6-tag, another histidine (H75), and a glutamate residue (E179) from a neighboring symmetry mate, forming a crystal contact.
Figure 3Crystal structure of CPR-C4. A, ribbon diagram of the CPR-C4 dimer (crystal form 1) showing the two molecules in the asymmetric unit perpendicular to the rotational noncrystallographic symmetry axis; chain A is shown in teal, chain B in crimson; Zn2+ ions are shown as gray spheres with 1.0 van der Waals radii. B, CPR-C4 monomer (form 1 chain A) orientated to highlight the position of the tri-aspartate Zn2+-binding site, indicated with a black box. The main chain is shown with ribbon representation; aspartate side chains are shown with cylinder representation, O atoms in red; Zn2+ ions are shown as gray spheres with 0.5 van der Waals radii; the coordinated water molecule is shown as a red sphere. C, close-up view of the Zn2+-binding site as indicated in (B), showing tetrahedral coordination to three aspartate side chains and a water molecule; distances in Å are indicated with dashed lines; aspartate side chains are shown with cylinder representation, O atoms in red; the Zn2+ ions are shown as gray spheres with 0.5 van der Waals radii; the coordinated water molecule is shown as a red sphere. D, least-squares superposition of CPR-C4 chains in the same orientation as (B): form 1 chain A shown in teal, form 1 chain B in crimson, form 2 in light blue, and form 3 chain A in white; Zn2+ ions found in form 1 chain A only have been omitted for clarity. E, least-squares superposition of the tri-aspartate Zn2+ site across the CPR-C4 chains colored as in (D); Zn2+ ions from form 1 chain A are shown as gray spheres with 0.5 van der Waals radii. CPR, Candidate Phyla Radiation.
The three structures in the different crystal forms superimpose very well, with no significant deviation (Fig. 3D and Table S1). The two chains of form 3 are very closely aligned, with a RMSD of only 0.27 Å. The two chains of form 1 are less closely aligned (RMSD 0.52 Å), likely due to slight differences resulting from Zn2+ binding to one chain. While form 2 has a single chain in the asymmetric unit, a symmetry dimer is observed with RMSD values of 0.37 Å and 0.31 Å against the form 1 and form 3 noncrystallographic dimers, respectively (Fig. S6). Despite the lack of Zn2+ in crystal forms 2 and 3, the key aspartate residues superimpose well across all three structures (Fig. 3E) and the geometry of the binding site is maintained in each structure. Figure 4 shows a comparison of electron density at this tri-aspartate site across the CPR-C4 crystal structures, demonstrating that Zn2+ is only present in crystal form 1, with no other metal cations binding in its absence. These results show that, although Zn2+ dramatically stabilizes the protein, its presence does not affect the overall structure and the protein adopts its native conformation even in the absence of Zn2+.
Figure 4Comparison of electron density at the tri-aspartate Zn2+-binding site between the CPR-C4 crystal forms. In all panels, the main chain is shown with ball and stick representation in teal, O atoms in red, N atoms in blue, S atoms in yellow; water molecules are shown as red spheres. The 2|Fo|-|Fc| map is shown in gray with cylinder representation at contour level σ = 1.5. This high sigma was chosen to make clear the position of Zn2+ and coordinating residues in (A). At σ = 1.0, D182 is well fitted in density across all three crystal forms. A, electron density for crystal form 1 chain A showing the location of bound Zn2+, depicted as a gray sphere, with coordinating water molecule and aspartate residues. B, electron density for form 1 chain B. C, electron density for form 2. D, electron density for form 3 chain A. CPR, Candidate Phyla Radiation.
The inherent inability to cultivate the source organism makes it difficult to confirm that Zn2+ is the native metal of the CPR-C4 tri-aspartate binding site. However, the absence of other metal cations in the zinc-free crystal structures implies this is not a promiscuous site, and the tetrahedral ligand arrangement is strongly preferable for Zn2+ binding over other transition metal cations or the sodium and potassium present in the buffers used (
IUCr
Metal–ligand geometry relevant to proteins and in proteins: Sodium and potassium.
). Zn2+ was also incorporated into the protein without being supplemented in expression media or buffers, and TSA showed vast protein stabilization upon the addition of Zn2+. This evidence all supports the presence of an intrinsic Zn2+-binding site in CPR-C4.
Bioinformatic structural analysis for determination of function
In order to determine the function of the novel CPR-C4 protein, 3DM (
) analysis was conducted to identify structural homologs for detailed comparison. Remarkably, two proteins from H. sapiens, tubulin tyrosine carboxypeptidases 1 and 2 (also known as vasohibins VASH1 and VASH2, respectively), were identified as sharing the core mixed αβ fold of CPR-C4 (Fig. 5A). These carboxypeptidases, bound to the α-helical small vasohibin binding protein, cleave the C-terminal tyrosine residue of α-tubulin as part of microtubule regulation, utilizing a noncanonical Cys-His-Leu(carbonyl) catalytic triad (
Wang N.
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Ryul Choi S.
Boulan B.
Peris L.
Olieric N.
Bao H.
Krichen F.
Andrieux A.
Olieric V.
Moutin M.-J.
Steinmetz M.O.
Huang H.
Structural basis of tubulin detyrosination by the vasohibin–SVBP enzyme complex.
Nat. Struct. Mol. Biol. 2019; 26: 571-582
Li F.
Qi S.
Luo X.
Yu H.
Structural basis of tubulin detyrosination by vasohibins.
Nieuwenhuis J.
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Bleijerveld O.B.
Mazouzi A.
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Celie P.
Altelaar M.
Knipscheer P.
Perrakis A.
Blomen V.A.
Brummelkamp T.R.
Vasohibins encode tubulin detyrosinating activity.
Slep K.C.
Cytoskeletal cryptography: Structure and mechanism of an eraser.
). These human isoforms are part of the transglutaminase-like cysteine protease superfamily and share a sequence identity of 87.8% with each other, though only show 17.6% (VASH1) and 15.4% (VASH2) sequence identity to CPR-C4.
Figure 5Sequence and structural analysis of CPR-C4 with 3DM. A, ribbon diagram showing the structural alignment of CPR-C4 (form 1 chain A, teal) with Homo sapiens VASH1 (6J8F chain B, green, RMSD 1.98 Å) and VASH2 (6J4P chain A, yellow green, RMSD 1.92 Å) through least-squares superposition. B, the 3DM core region, sharing significant structural similarity across CPR-C4 and human VASH1/2, is shown in the same color representation as (A); the remaining 'variable regions' are shown in dark gray for CPR-C4 and in light gray for VASH1/2. C, amino acid sequence of CPR-C4, with the 3DM core region underlined. Residues conserved in ≥95% of 3DM superfamily members are shown in blue; residues that form ≥8 ligand contacts in the superfamily are shown in green. The three residues boxed in red form the conserved catalytic triad (C61, H93, L115, following CPR-C4 sequence numbering). The reside boxed in purple is the conserved tyrosine involved in Leu(carbonyl) positioning in VASH1/2 (
). CPR, Candidate Phyla Radiation.
To further investigate the evolutionary relationship between these proteins, the 3DM system for CPR-C4 was subsequently extended to include the two additional subfamilies of the newly identified human VASH1/2 structural homologs. These subfamilies were denoted 6J8FB and 6J4PA after their template structure Protein Data Bank accession codes (
). The resulting 3DM superfamily system contains 657 and 817 sequences homologous to the human VASH1 and VASH2 proteins, respectively.
The 3DM alignment tool (
) was then used to identify highly conserved residues in the superfamily as well as residues known to form ligand contacts in structures collated in the 3DM system. One hundred thirty of 221 residues in the CPR-C4 sequence fall within the structurally conserved core region for the extended superfamily, as shown in Figure 5, B and C. Also within this region is the known substrate-binding site of the vasohibins (
): of the 17 residue positions in VASH1/2 identified to form ligand contacts, 15 positions were found in the core region shared with CPR-C4. Only five of the 15 residue identities were conserved in the CPR-C4 sequence; however, three of these importantly comprise the known catalytic triad of VASH1/2 required for protease activity: C61, H93, and L115 (following the CPR-C4 sequence) (Fig. 6A). This unconventional catalytic triad is highly conserved throughout all three subfamilies (Fig. 6B), and the residues structurally superpose well across the three template structures (Fig. 6C). C61 and H93 are almost entirely conserved in both the CP01A subfamily and the extended 3DM superfamily, and leucine is the dominant residue identity at position 115. C61, H93, and L115 also form nine, 10, and eight ligand contacts in the superfamily, respectively, highlighting their importance for ligand binding in VASH1/2.
Figure 6Analysis of the CPR-C4 catalytic triad. A, close-up view of the catalytic triad residues in the CPR-C4 crystal structure (form 1 chain A). The main chain is shown with ribbon representation in teal; side chains of the catalytic triad residues and the backbone carbonyl of L115 are shown with stick representation, O atom in red, N atoms in blue, S atom in yellow; interatomic distances in Å are indicated with dashed lines. The average B-factors for these residues are 92.9 Å2, 89.4 Å2, and 84.9 Å2 for C61, H93, and L115, respectively. B, conservation of the catalytic triad residue identities across the CP01A subfamily and the extended superfamily comprising the CP01A, 6J4PA, and 6J8FB subfamilies; ∗ indicates all other residue identities. C, catalytic triad residues in CPR-C4 (form 1 chain A, teal) aligned with equivalent 3D residues from human VASH1 (6J8F chain B, green) and VASH2 (6J4P chain A, yellow green) through least-squares superposition; the main chain is shown with ribbon representation; catalytic triad side chains and the backbone carbonyl group of L115 are shown with cylinder representation, O atoms in red, N atoms in blue, S atoms in yellow. D, electrostatic surface representation of CPR-C4 (form 1 chain A) showing the proposed substrate-binding pocket of CPR-C4, lined with positively charged residues; red shows positive potential, blue shows negative potential; the surfaces of the catalytic triad residues are shown in yellow; Zn2+ ion represented as a gray sphere with 1.0 van der Waals radius. CPR, Candidate Phyla Radiation.
The occurrence of leucine makes this an atypical catalytic triad for a cysteine protease. Based on the mechanism suggested for the vasohibins, it was proposed that the backbone carbonyl of L115 in CPR-C4 forms a hydrogen bond (H-bond) with the imidazole ring of H93, which in turn activates the cysteine thiol. The tyrosine residue known to help position the key leucine carbonyl for H-bonding with His in the human vasohibins is also present in CPR-C4, with 92.5% conservation in the CP01A subfamily and 95.8% conservation in the extended superfamily (Fig. S7). Intriguingly, a serine residue (S108) was also identified in close proximity to H93, at a distance appropriate for H-bonding (2.8 Å), which could play an additional role in activation for catalysis. Nonetheless, Ser is only present in 41.9% of CP01A subfamily sequences, with Ala in over 50% (473/938) and Gly in all vasohibin family sequences. This serine residue is therefore unlikely to be crucial to function, though may play a role in some CP01A subfamily proteins.
While the conservation of the unusual catalytic triad in both sequence and structure is a crucial indicator of shared function, there are key differences between CPR-C4 and the vasohibins. Unlike VASH1/2, which are known to require a small binding protein, there is no indication that CPR-C4 requires a binding partner for catalysis. CPR-C4 is a homodimer and lacks the helices that are known to form intermolecular contacts between monomeric VASH1/2 and the small vasohibin binding protein. In the vasohibin structures, the conserved catalytic triad is also located deep in a negatively charged pocket, known to be the substrate binding pocket for α-tubulin (
). In CPR-C4, the pocket containing the proposed C61-H93-L115 catalytic triad is formed of positively charged residues (Fig. 6D), implying a negatively charged substrate. This is in contrast to the positively charged α-tubulin substrate of VASH1/2, which is also crucially lacking in bacteria. The structure-based multiple sequence alignment analysis therefore points to CPR-C4 functioning as a cysteine protease, utilizing the same noncanonical catalytic triad as the VASH1/2 structural homologs, albeit with a very different substrate.
Analysis of the Zn2+-binding site
To evaluate the role of Zn2+, the extent of aspartate conservation at the binding site residue positions was assessed across the CP01A subfamily and the wider superfamily (Fig. 7A). All three positions are in the core structural region conserved between the subfamilies: following the CPR-C4 sequence, these positions are denoted 83, 92, and 182. There is considerable conservation in the CP01A subfamily, with aspartate being the predominant residue at all three positions. However, less than half of the sequences contain a residue aligned at position 182. The tri-aspartate Zn2+-binding site is therefore not a requirement for proteins in the CP01A subfamily.
Figure 7Analysis of the CPR-C4 tri-aspartate Zn2+-binding site. A, conservation of equivalent residues of the CPR-C4 Zn2+-binding site across the CP01A subfamily and the extended superfamily containing the CP01A, 6J4PA, and 6J8FB subfamilies; ∗ indicates all other residue identities. B, Zn2+-binding site from the CPR-C4 structure (form 1 chain A, teal) aligned with equivalent residues in human VASH1 (6J8F chain B, green) and VASH2 (6J4P chain A, yellow green) through least-squares superposition; the main chain is shown with ribbon representation; residue side chains are shown with cylinder representation, O atoms in red, N atoms in blue; Zn2+ ions are shown as a gray spheres with 0.5 van der Waals radii and the coordinated water molecule is shown as a red sphere (note: these are only present in the CPR-C4 form 1 chain A structure). CPR, Candidate Phyla Radiation,
The conservation of aspartate seen in the CP01A subfamily also does not extend to the wider superfamily. All instances of aspartate at these positions in the superfamily occur in sequences from the CP01A subfamily, with none in 6J8FB or 6J4PA subfamily sequences. At position 83, the predominant residue in the superfamily is serine, occurring in 63.4% of sequences. At position 92, there are similar proportions of aspartate, histidine, and arginine, and tryptophan is overwhelmingly the most common residue at position 182 (77.0%). Throughout the 6J4PA or 6J8FB subfamilies, there are also no residues known to form metal ion contacts. Residues 83, 92, and 182 in CPR-C4, and equivalent residues in VASH1/2, are superposed in Figure 7B. While 83 and 92 superpose well across the structures, the coordination of D182 to Zn2+ in CPR-C4 pulls the chain out of alignment with the vasohibin structures at the start of the α3 helix. This evidence collectively points to a lack of conservation of the CPR-C4 Zn2+-binding site in the vasohibin subfamilies.
Protease activity of CPR-C4
The structure-based multiple sequence alignment analysis strongly implied that CPR-C4 is a protease, utilizing a cysteine-based catalytic triad to cleave an unidentified substrate. The enzyme is expected to be highly specific based on the unusual active site geometry and what is known of the VASH homologs. In order to confirm protease activity, biochemical assays were conducted using a casein derivative substrate labeled with green fluorescent BODIPY-FL dye (
Thompson V.F.
Saldaña S.
Cong J.
Goll D.E.
A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases.
Anal. Biochem. 2000; 279: 170-178
), as a substitute for the unknown substrate. Highly purified protein (Fig. S2A) was used in these assays to minimize the likelihood of contamination. As shown in Figure 8A, the addition of increasing concentrations of CPR-C4 to a fixed amount of casein substrate resulted in a rise in fluorescence emission at 528 ± 20 nm after excitation at 485 ± 20 nm. Additions of CPR-C4 in the nM range (to a maximum of 0.05 μM) resulted in a significant increase in fluorescence relative to nonproteolytic controls. This indicates the presence of fluorescently labeled peptide fragments due to proteolytic cleavage of the BODIPY-FL casein substrate by CPR-C4. The time course shown in Figure 8B also shows that, at a fixed concentration of CPR-C4 (0.025 μM), the fluorescence intensity increases over time as the BODIPY-FL casein substrate is cleaved to fluorescent fragments by CPR-C4. This experimental data confirms that CPR-C4 shows protease activity, as was projected from the structure-based multiple sequence alignments.
Figure 8Experimental analysis of CPR-C4 protease activity. A, protease activity data for increasing concentrations of CPR-C4 in the presence (n = 8, ± st. dev.) and absence (n = 3, ± st. dev.) of BODIPY-FL casein substrate, measured as fluorescence intensity in relative fluorescence units (RFU) after 3 h at 30 °C. B, time course of the increase in fluorescence intensity for 0.025 μM CPR-C4 in the presence (n = 8, ± st. dev.) and absence (n = 3, ± st. dev.) of BODIPY-FL casein substrate. CPR, Candidate Phyla Radiation.
In order to analyze the evolutionary relationship between CPR-C4 and the human VASH proteins in greater detail, a phylogenetic tree was generated from 92 protein sequences with significant similarity to CPR-C4 and VASH1/2 from a broad range of taxa (Fig. S8). These sequences are based on separate BLAST (
) searches of the CPR-C4 and VASH1/2 protein sequences using an E value cut-off of 0.05. After multiple sequence alignment with ClustalW (
Sievers F.
Wilm A.
Dineen D.
Gibson T.J.
Karplus K.
Li W.
Lopez R.
McWilliam H.
Remmert M.
Söding J.
Thompson J.D.
Higgins D.G.
Fast, scalable generation of high-quality protein multiple sequence alignments using clustal Omega.
Mol. Syst. Biol. 2011; 7: 539
), the phylogenetic tree was constructed using MEGA X (
Stecher G.
Li M.
Knyaz C.
Tamura K.
MEGA X: Molecular evolutionary genetics analysis across computing platforms.
Mol. Biol. Evol. 2018; 35: 1547-1549
) with maximum-likelihood methods. Bootstrap tests were also applied to assess the confidence.
The tree is divided almost perfectly in two, with sequences relating to human VASH1/2 congregating in the top half, grouped into animalia and fungi species in light and dark blue, and Plantae species in green. Sequences from Firmicutes bacteria were the only prokaryotic sequences identified with similarity to human VASH1/2 in the original BLAST (
) search. The lower half of the tree is comprised of bacterial (non-CPR and CPR in yellow and light yellow, respectively), archaeal (in red), and algal (in orange) protein sequences identified by BLAST (
) on CPR-C4. Importantly, no eukaryotic sequences were identified in this search. The taxa at the node linking these two groups of sequences only clustered together in 195/500 repeats (39%, Fig. S8). This implies a weak, if any, evolutionary relationship between CPR-C4 and the human vasohibins that is not identifiable by sequence analysis alone.
Validation of the CPR-C4 ORF gene product
While a consistent feature of the CPR is small genomes, the CPR-C4 contig is significantly smaller (0.4 Mbp) than previously identified closed genomes from the CPR (0.7–1.2 Mbp) (
), suggesting it does not cover an entire genome. Due to their small size, CPR genomes could be expected to have a higher percentage of genes with known functions than usual, dominated by enzymes with near universal functions crucial for survival. However, less than one third of the genes comprising the CPR-C4 contig could be assigned predicted functions based on sequence analysis, compared to up to 70% of a typical newly sequenced bacterial genome (
Bork P.
Powers and pitfalls in sequence analysis: The 70% hurdle.
Genome Res. 2000; 10: 398-400
). Since the unearthing of the CPR within the tree of life is still recent, it is unsurprising that such genomes remain poorly understood and contain large proportions of uncharacterized gene products. Many of the hypothetical proteins identified in the CP01A subfamily with high sequence similarity to CPR-C4 are also from candidate phyla bacteria, indicating that this protein is highly conserved and therefore important within the CPR.
Thermostability
Considering that the source organism of CPR-C4 originates from a sample collected at 75 °C, it is perhaps surprising that the Tm of CPR-C4 without additives is so low (Fig. 2B). While it may be expected that proteins originating from thermophilic organisms would be intrinsically thermostable, this is not guaranteed. The protein environment in vivo is often responsible for a large amount of stabilization, including through molecular chaperones and compatible solutes (
Ladenstein R.
Proteins from hyperthermophiles: Stability and enzymatic catalysis close to the boiling point of water.
Adv. Biochem. Eng. Biotechnol. 1998; 61: 37-85
). CPR-C4 was indeed stabilized by a wide variety of salts at high concentrations (Fig. 2A), which provides an insight into how thermotolerance is achieved in its natural environment.
Role of Zn2+ in CPR-C4
Zn2+ ions in proteins can be structural or participate directly in enzyme function (
Mccall K.A.
Huang C.-C.
Fierke C.A.
Function and mechanism of zinc metalloenzymes.
J. Nutr. 2000; 130: 1437-1446
). The nature of the CPR-C4 Zn site is at first glance indicative of a catalytic over a structural Zn site, where the metal coordination sphere is often comprised of protein side chains only (
Auld D.S.
Zinc coordination sphere in biochemical zinc sites.
Biometals. 2001; 14: 271-313
Andreini C.
Bertini I.
A bioinformatics view of zinc enzymes.
J. Inorg. Biochem. 2012; 111: 150-156
). However, the arrangement and identities of the residues forming this metal binding site are unusual. In classic catalytic Zn metalloproteins, two of the protein ligands (L1 and L2) are typically in close proximity in the sequence, that is, one to three amino acids apart, with much greater variation in the distance of the third coordinating residue (L3) along the chain (
). In the case of CPR-C4, the first two ligands (D83) and L2 (D92) are separated by eight residues, and L3 (D182) is well removed from the first two ligands (90 residues). The tri-aspartate arrangement is also atypical, with histidine accounting for the majority of residues coordinated to Zn2+ in catalytic sites of Zn metalloproteins and cysteine for structural sites (
). Considering the metal coordination alone, CPR-C4 therefore appears to contain an unusual but possible catalytic Zn2+ site of unknown function. However, since Zn2+ or other metal ions are not required for protease activity in the vasohibins, it is unlikely that the Zn2+ in CPR-C4 has a catalytic role or is a requirement for activity. The TSA experiments show that Zn2+ instead plays a stabilizing structural role, increasing the thermotolerance of the protein significantly.
From structure to function
The novel crystal structure of CPR-C4 demonstrates the power of structure determination for unraveling the function of hypothetical proteins for which sequence alone is insufficient to assign a putative function. For this purpose, the 3DM (
) information system was an invaluable in silico screen for predicting the experimentally confirmed protease activity of CPR-C4, which can now be applied to the hundreds of other hypothetical proteins sharing extremely high sequence similarity. The remote relationships between such sequences, coupled with structural similarities, are also key to the success of very recent structure prediction tools like AlphaFold2 and RoseTTAFold (
Jumper J.
Evans R.
Pritzel A.
Green T.
Figurnov M.
Ronneberger O.
Tunyasuvunakool K.
Bates R.
Žídek A.
Potapenko A.
Bridgland A.
Meyer C.
Kohl S.A.A.
Ballard A.J.
Cowie A.
Highly accurate protein structure prediction with AlphaFold.
Cramer P.
AlphaFold2 and the future of structural biology.
Baek M.
DiMaio F.
Anishchenko I.
Dauparas J.
Ovchinnikov S.
Lee G.R.
Wang J.
Cong Q.
Kinch L.N.
Schaeffer R.D.
Millán C.
Park H.
Adams C.
Glassman C.R.
DeGiovanni A.
Accurate prediction of protein structures and interactions using a three-track neural network.
). Both tools were able to generate models of high enough quality for CPR-C4 crystal structure determination by molecular replacement methods (Fig. S9), demonstrating their potential for aiding experimental structure solutions of other hypothetical proteins within the CPR and beyond.
The structure-based multiple sequence alignment methods used in this work result in better quality alignments than classical multiple sequence alignments, and here enabled detailed comparisons of structurally equivalent residues between the homologous CPR-C4 and human VASH1/2 structures, despite the low sequence identity. The conservation of the unusual vasohibin catalytic triad, not only in the CPR-C4 structure, but throughout the CP01A subfamily, suggests that these three residues are also key to the function of CPR-C4 and its homologs throughout the CPR. The structure-based sequence alignments indicate that CPR-C4 is also a cysteine protease, utilizing the same general mechanism and catalytic triad as the VASH1/2 structural homologs. The occurrence of leucine makes this an atypical catalytic triad, and the ability of other amino acids to fulfill its role explains why it is less well conserved than its essential cysteine and histidine counterparts. The distance between the imidazole ring of H93 and carbonyl of L115 in CPR-C4 is larger than in the vasohibins but is still within the acceptable range for H-bonding. Furthermore, these side chain conformations are also dynamic and will change during catalysis.
Evolutionary relationship between CPR-C4 and human VASH1/2
The discovery of a close structural relationship between CPR-C4 and the human VASH proteins was unexpected, particularly when considering the low sequence identity. While our phylogenetic analysis did not enable identification of a common ancestor for CPR-C4 and VASH1/2, it may suggest a possible, albeit weak, evolutionary link between these proteins. This notion is strongly supported by the overall structural and functional resemblances between the proteins. In particular, the extremely unusual VASH1/2 catalytic triad is conserved in CPR-C4 and its prokaryotic homologs, both in sequence and space. These factors strongly suggest that the proteins are indeed related by divergent evolution, pointing to an important cysteine protease present in species from across the tree of life.
Potential functional role of CPR-C4 in CPR bacteria
It is well understood that species within the CPR are symbionts, with most having little capability for the de novo synthesis of many essential metabolites, including amino acids. However, little is currently known about their interactions with other taxa. A few species are symbiotic with eukaryotes (
Gong J.
Qing Y.
Guo X.
Warren A.
"Candidatus Sonnebornia yantaiensis", a member of candidate division OD1, as intracellular bacteria of the ciliated protist Paramecium bursaria (Ciliophora, Oligohymenophorea).
Syst. Appl. Microbiol. 2014; 37: 35-41
), though the abundance and diversity seen in samples with minimal eukaryotic organisms suggests most are likely to be associated with bacteria or archaea. It is probable that the CPR-C4 source organism has a bacterial symbiotic partner, since the vast majority (72.2%) of the 442,373 sequenced genes from the metagenome was bacterial, with only 5.3% archaeal genes, 1.7% eukaryotic genes, and 20.6% from unknown or uncharacterized organisms, alongside 0.2% from viruses.
The CPR-C4 gene appears unrelated to its neighboring genes on the contig (Fig. 1A), which makes speculating on a potential function in vivo more challenging. The degradation of peptide substrates by CPR-C4 could be necessary to provide essential amino acid metabolites both to the CPR organism and its symbiotic partner. CPR genomes are enriched with pili-related genes compared to other bacteria, and pili-like structures have been observed on the surfaces of CPR cells, which could provide a mechanism for metabolite uptake (
Dick G.J.
Hauser L.J.
Hyatt D.
Dill B.D.
Land M.L.
Enigmatic, ultrasmall, uncultivated Archaea.
Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 8806-8811
). CPR-C4 could also be involved in the maintenance or regulation of these pili structures.
A novel protein from metagenomics
This work has identified a novel protease from the CPR and presents the first crystal structure of a protein from a thermophilic CPR bacterium. In the process, a family of novel bacterial proteases featuring an unusual catalytic triad, with hundreds of members across the CPR, has been identified. Roughly 60% of market enzymes worldwide are proteases, with applications throughout several important sectors (
Razzaq A.
Shamsi S.
Ali A.
Ali Q.
Sajjad M.
Malik A.
Ashraf M.
Microbial proteases applications.
Front. Bioeng. Biotechnol. 2019; https://doi.org/10.3389/fbioe.2019.00110
). This work is a significant starting point in the discovery of applications for this unusual cysteine protease, with its potential thermophilic properties making it an interesting candidate for biotechnological applications in future.
A significant hurdle to studying species within the CPR is their inability to be cultured using standard methods. The metagenomic approach, which removes the need for cultivation, has greatly expanded our understanding of microbial life through the discovery of previously inaccessible genomes, such as that containing the CPR-C4 gene (
). With the progression of phylogenetic methods, and an increase in available genome sequences, it is likely that the roles CPR species play in the biosphere, and the extent to which they dominate the bacterial domain, will become clearer in the near future. Many hypothetical proteins with little or no annotation will be recognized to complete metabolic pathways that currently appear broken or missing completely from CPR genomes (
). Isolation and sequencing of additional genomes from a wider variety of sources is necessary to further explore the radiation at the biochemical and structural levels through the emergence of conserved protein families. The fact that roughly 70% of the genes on the CPR-C4 contig have been annotated as hypothetical proteins highlights how poorly understood these genomes are, and the vast inventories of proteins with potentially novel functions that they contain make them enticing targets for further exploration. The recurrence of CPR-C4–type proteases with high sequence conservation throughout the candidate phyla hints at an important function in vivo that cannot be confirmed without greater understanding of the lifestyle of CPR organisms.
Target selection and annotation
The biodiscovery pipeline has previously been outlined in detail (
). Briefly, samples collected from natural environments were processed with consecutive steps of microfiltration, concentration, and DNA extraction in order to isolate genetic material for total metagenomic DNA analysis by next generation sequencing platforms (Illumina MiSeq, HiSeq; Oxford Nanopore). The output from sequencing was assembled and binned for downstream analysis, including quality control and assembly into longer contigs, followed by gene prediction, taxonomic and functional annotation of predicted genes, and binning into metagenome-assembled genomes. Gene products with high innovation potential were selected for expression and characterization, prioritizing those showing extended conservation across genomes but with unknown function.
Cloning, protein production, and characterization
Cloning of the CPR-C4 gene into the pJOE5751.1 vector (
Wegerer A.
Sun T.
Optimization of an E. coli L-rhamnose-inducible expression vector: Test of various genetic module combinations.
BMC Biotechnol. 2008; 8: 2
) and pET28a(+)TEV was conducted by Genscript: the target DNA fragment was inserted via BamHI/BsrGI restriction sites to form the pJOE5751.1-CPRC4 construct and via BamH1/Xho1 restriction sites for generation of the pET28a(+)TEV-CPRC4 construct. Amino acid sequences of the fusion proteins expressed using these constructs are detailed in Fig. S4.
After separate transformations of these constructs into T7 Express Strain competent E. coli cells (New England Biolabs), 25 ml precultures were grown overnight in LB at 37 °C (pJOE5751.1-CPRC4 with 100 μg/ml ampicillin; pET28a(+)TEV-CPRC4 with 50 μg/ml kanamycin). One liter LB cultures with appropriate antibiotic were inoculated with the preculture and grown (37 °C, 150 rpm) until an OD600 of 0.5 was reached, at which point overexpression was induced with the addition of L-rhamnose (0.2%, pJOE5751.1-CPRC4) or IPTG (1 mM, pET28a(+)TEV-CPRC4) at 25 °C. The cultures were incubated with shaking overnight (25 °C, 150 rpm) and subsequently centrifuged at 1300g for 25 min at 4 °C. Cell pellets were resuspended in 20 mM Hepes pH 7.4, 300 mM NaCl, 40 mM imidazole (pJOE5751.1-CPRC4), or 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 40 mM imidazole (pET28a(+)TEV-CPRC4), with cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche), then sonicated and centrifuged at 50,000g for 50 min at 4 °C. The supernatant was filtered (0.2 μm) and loaded onto a 1 ml HisTrap HP affinity column (Cytiva) for an imidazole gradient fast protein liquid chromatography separation (40 mM to 500 mM imidazole) using an ÄKTA pure. Protein from the pJOE5751.1-CPRC4 construct was subsequently incubated overnight at 4 °C with TEV protease for His6-tag removal and repurified by imidazole gradient fast protein liquid chromatography separation. CPR-C4 protein molecular weights were verified by electrospray ionization time-of-flight mass spectrometry and SDS-PAGE prior to crystallization and TSA experiments.
Subsequent expression experiments (pJOE5751.1-CPRC4) included addition of ZnCl2 (100 μM in culture) to LB media for 25 ml precultures and 1 l cultures. Purification by imidazole gradient fast protein liquid chromatography separation was also improved with the use of sodium phosphate buffers (20 mM Na phosphate pH 7.4, 500 mM NaCl, 40 mM to 1 M imidazole gradient).
Size-exclusion analysis was conducted using a HiLoad 16/600 Superdex 75 pg column calibrated using the LMW Calibration Kit (Cytiva) according to instructions provided by the manufacturer. One milliliter of protein sample was injected onto the pre-equilibrated column and was eluted from the column using 20 mM Na phosphate pH 7.4, 500 mM NaCl.
TSA was conducted using the Durham Salt and pH Screens (Molecular Dimensions). Protein samples were dialyzed into 10 mM sodium phosphate pH 7.4, 100 mM NaCl. SYPRO orange dye (4 μl, 5000× in DMSO) was added to the protein sample (1 ml, 0.8 mg/ml) to give a protein-plus-dye solution. Ten microliters of Durham Screen condition was added to 10 μl of the protein-plus-dye solution in each well of a standard 96-well PCR plate, which was sealed with thermostable film before centrifugation (2 min, 160g). The temperature of the plate was held for 1 min at 1 °C intervals from 24 °C to 96 °C, and fluorescence data for melt temperature, Tm, experiments were collected with an Applied Biosystems 7500 Fast Real-Time PCR System, with an excitation range of 540 to 550 nm. Data were analyzed with in-house Microsoft Excel scripts and the graphical user interface-based python program NAMI (
) using the emission signal at 567 to 596 nm. Three technical repeats were conducted, with SD used to report experimental variability.
Purified protein from the pJOE5751.1-CPRC4 construct, expressed without the addition of ZnCl2 to media, was dialyzed into SGC buffer (20 mM Hepes pH 7.4, 300 mM NaCl, 10% v/v glycerol). Initial high-throughput screening was conducted using a Mosquito Crystal protein robot (STP Labtech) and a range of commercially available crystallization screens (Molecular Dimensions). Eighty microliters crystallization reagent was added to reservoirs in MRC 96-well sitting drop plates (Jena Biosciences). Screen conditions and protein samples were combined in two ratios for each screen: 1:1 (100 nl protein: 100 nl crystallization reagent) and 2:1 (200 nl protein: 100 nl crystallization reagent). Plates were observed periodically for crystal formation using a Leica MZ16 Stereomicroscope. Manual crystallization experiments were conducted using the hanging drop vapor diffusion method with 500 μl crystallization reagent in reservoirs and ratios of 1:1 (1 μl: 1 μl) and 2:1 (2 μl: 1 μl) protein solution to crystallization reagent.
Crystal form 1 (dodecahedrons) was obtained at 20 °C using a protein solution of 4 mg/ml in SGC buffer across 25% (24 of 96 conditions) of the Morpheus HT-96 screen and was reproducible in further manual experiments with the Morpheus screen. Crystals predominantly formed in conditions containing a pH 6.5 MES monohydrate buffer, with mixtures of carboxylic acids, amino acids, or salt additives as present in the Morpheus screen. These crystals were fragile and frequently attached to the well surfaces of sitting drop trays. The hanging drop vapor diffusion method was therefore used to make manipulation of the crystals more practicable. Crystals leading to the structural solution of CPR-C4 were grown in 0.09 M NPS, 0.1 M buffer system 1 pH 6.5, 50% v/v precipitant mix 2 from the Morpheus HT-96 screen (Molecular Dimensions).
Crystal form 2 (cubic rods) were grown at 20 °C using 2.4 M sodium malonate dibasic monohydrate pH 7.0 (condition F9 from the JCSG-plus HT-96 screen (Molecular Dimensions)). These crystals were more robust and mountable directly from 96-well sitting drop trays.
Crystal form 3 was generated using protein from the pET28a(+)TEV-CPRC4 construct designed to allow removal of the N-terminal His-tag. Purified protein (after His6-tag removal with TEV protease) was dialyzed into phosphate buffer (20 mM sodium phosphate pH 7.4, 500 mM NaCl) for high-throughput crystallization screening with a variety of commercial screens (Molecular Dimensions). Crystals were obtained from a 6 mg/ml protein solution at 20 °C in 0.2 M lithium sulfate, 0.1 M Tris pH 8.5, 15% w/v PEG 4000.
Data collection, structure solution, and refinement
All crystals were cryoprotected using a 1:1 ratio of glycerol to crystallization solution and flash frozen in liquid nitrogen. Data were collected remotely using beamlines I03 (form 1), I24 (form 2), and I04 (form 3) at the Diamond Light Source. In the case of crystal form 1, native diffraction data were initially processed using autoPROC (
) with STARANISO (
) via the ISPyB pipeline, and the autoprocessed data were imported into CCP4i2 (
Ballard C.C.
Cowtan K.D.
Dodson E.J.
Evans P.R.
Keegan R.M.
Krissinel E.B.
Leslie A.G.W.
McCoy A.
McNicholas S.J.
Potterton E.A.
Powell H.R.
Overview of the CCP4 suite and current developments.
). Data were phased by multiple wavelength anomalous diffraction at the Zn edge (1.2824 Å, identified with an X-ray absorption edge scan; Fig. S5) and native (0.9763 Å) wavelengths, using the SHELXC/D/E (
) via the CRANK2 pipeline (
Waterreus W.-J.
Sikharulidze I.
Abrahams J.P.
de Graaff R.A.G.
Recent advances in the CRANK software suite for experimental phasing.
). Peak data were subsequently reprocessed in XDS (
Kabsch W.
XDS.
). Rotational noncrystallographic symmetry was identified between two protein chains in the asymmetric unit; this was exploited through density modification with Parrot (
Recent developments in classical density modification.
) applying local noncrystallographic symmetry restraints (
Usón I.
Schneider T.R.
Dauter Z.
Fritz H.-J.
1.7 Å structure of the stabilized REIv mutant T39K. Application of local NCS restraints.
) and a solvent content of 69%, corresponding to two protein molecules in the asymmetric unit. Chain A of the model was built using iterative rounds of manual modifications in Coot (
Lohkamp B.
Scott W.G.
Features and development of Coot.
Acta Cryst. 2010; 66: 486-501
) and REFMAC5 (
) refinement cycles and used to fit chain B by molecular replacement with PHASER (
Grosse-Kunstleve R.W.
Adams P.D.
Storoni L.C.
Phaser crystallographic software.
J. Appl. Crystallogr. 2007; 40: 658-674
). The CPR-C4 model was refined using Coot (
) with jelly-body restraints for initial rounds of refinement and using local noncrystallographic symmetry restraints (
Diffraction data for CPR-C4 crystal forms 2 and 3 were processed using xia2 Dials DUI (
Winter G.
Waterman D.G.
Parkhurst J.M.
Brewster A.S.
Gildea R.J.
Gerstel M.
Fuentes-Montero L.
Vollmar M.
Michels-Clark T.
Young I.D.
Sauter N.K.
Evans G.
DIALS: Implementation and evaluation of a new integration package.
Res. Pap. Acta Cryst. 2018; 74: 85-97
) and XDS (
), respectively. The structures were solved by molecular replacement with PHASER (
) using the structure of chain A from crystal form 1 as a homology model. The resulting structures were refined against the density using REFMAC5 (
) with jelly-body restraints for initial rounds of refinement and local noncrystallographic symmetry restraints (
) for crystal form 3, with manual adjustments made in Coot (
All model building and evaluation was performed with Coot (
). The final models were checked using MolProbity (
Arendall W.B.
Headd J.J.
Keedy D.A.
Immormino R.M.
Kapral G.J.
Murray L.W.
Richardson J.S.
Richardson D.C.
MolProbity: All-atom structure validation for macromolecular crystallography.
). Further crystallographic data are summarized in Table 1. Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 7OB6 (form 1), 7OB7 (form 2), and 7PJO (form 3). Ribbon diagrams were generated using CCP4mg (
McNicholas S.
Potterton E.
Wilson K.S.
Noble M.E.M.
Presenting your structures: The CCP4mg molecular-graphics software.
). Protein–protein interfaces were analyzed using PISA (
3DM analysis
A 3DM system (
) for the extended protein superfamily containing CPR-C4 and structural homologs was created using the amino acid sequence and .pdb file of the refined model (form 1 chain A). Two vasohibin structures (VASH1 and VASH2) were identified as sharing structural homology with CPR-C4 through structure superpositions. A structure-based multiple sequence alignment was used to determine conserved 'core' regions between these vasohibin structures and CPR-C4, followed by a BLAST (
) search against the three protein sequences resulting in a superfamily multiple sequence alignment containing 2441 sequences in total, including 46 identified during the Virus-X project (1.9%). A synchronized numbering system was used to assign all structurally equivalent residues in the 3DM system the same number (3D number) for direct comparison of corresponding residues between sequences, linking sequence alignment data to the template structures (H. sapiens VASH1/2 and CPR-C4 form 1). Many types of protein-related data, including protein-ligand contacts, residue mutations, and protein variants, were extracted from sequences, structures, and the accompanying literature, and collated in the 3DM system. Yasara (
Krieger E.
YASARA View - molecular graphics for all devices - from smartphones to workstations.
Bioinformatics. 2014; 30: 2981-2982
) was used to visualize structural alignment outputs from 3DM.
Protease activity assays
Protease activity assays were conducted using the commercially available Molecular Probes' EnzChek Protease Assay Kit (Invitrogen) with green fluorescence from BODIPY-FL casein (
). Purified CPR-C4 was dialyzed into 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 10 mM TCEP. Assays were conducted in a 96-well plate format in Corning nonbinding surface black fluorescence plates using a total reaction volume of 200 μl per well. Hundred microliters of BODIPY-FL casein substrate (10 μg/ml in 20 mM sodium phosphate pH 7.4, 500 mM NaCl) was added to 100 μl protein (concentrations ranging from 0 to 0.05 μM) before centrifugation (2 min, 160g). The plates were incubated at 30 °C and fluorescence was read using a Synergy HTX plate reader with a fluorescein filter (ex/em = 485 ± 20 nm/528 ± 20 nm; gain 100). Experiments were carried out with controls for background fluorescence taken in triplicate at each protein concentration without the BODIPY-FL casein substrate in order to account for any intrinsic fluorescent effects of the CPR-C4 protein. Eight technical repeats were conducted at each concentration of CPR-C4. Experimental variability is reported as SD.
Separate BLAST (
) searches were run with the CPR-C4 and H. sapiens VASH1/2 sequences using an E value threshold of 0.05. Ninety two representative protein sequences were selected from the results, covering a wide range of taxa and E values, and were aligned using ClustalW (
): 49 from the CPR-C4 search and 43 from VASH1/2 searches. The alignment was used to generate a phylogenetic tree using the Maximum Likelihood method and Jones-Taylor-Thornton matrix-based model in MEGA X (
Jones D.T.
Taylor W.R.
Thornton J.M.
The rapid generation of mutation data matrices from protein sequences.
Comput. Appl. Biosci. 1992; 8: 275-282
Felsenstein J.
Confidence limits on phylogenies: An approach using the bootstrap.
Evolution. 1985; 39: 783-791
Hillis D.M.
Bull J.J.
An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis.
Syst. Biol. 1993; 42: 182-192
). Five hundred replicates were used to calculate bootstrap values and used to establish the strength of the consensus tree. National Center for Biotechnology Information accession codes for all sequences can be found in Table S2. Evolutionary analyses were conducted in MEGA X (
Metagenomic data and the 3DM systems for CPR-C4 are available upon request. All other data needed to evaluate the conclusions in the paper are present in the article and/or the supporting information. The crystal structures of CPR-C4 have been deposited in the Protein Data Bank under accession codes 7OB6 (form 1), 7OB7 (form 2), and 7PJO (form 3).
We would like to thank all members of the Horizon 2020 Virus-X consortium for their collaboration. We are grateful to O. H. Fridjonsson, E. E. Gudmundsdottir, and G. Hreggvidsson for their work on bioprospecting and metagenome sequencing, and J. Kalinowski and A. Szerba for proving the database as part of the consortium. Thanks to S. Freitag-Pohl and I. Edwards for their laboratory assistance, C. Tomlinson for helpful crystallography discussions, and P. Denny for his guidance on phylogenetics. Thanks also to B. Vroling and Bio-Prodict for their general support. Thanks, as always, to Diamond Light Source and its staff for their wonderful facilities and assistance. K. A. S. C. thanks the EPSRC DTA for their support (EP/R513039/1).
K. A. S. C., A. A., and E. P. conceptualization; K. A. S. C., J. L., and E. P. methodology; K. A. S. C. investigation; A. A. and E. P. supervision; K. A. S. C. writing–original draft; K. A. S. C., J. L., A. A., and E. P. writing–review and editing.
European Research Council, European Union's Horizon 2020 research and innovation program grant 685778 (K. A. S. C., J. L., A. A., and E. P.).
Supplemental Figures S1–S9, Tables S1 and S2
Received in revised form: April 4, 2022
Received: October 14, 2021
Edited by Wolfgang Peti
Crown Copyright © 2022 Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.


1. I am a woman (35) suffering from cough for the three months. The problem seems to aggravate in my office.
Ans- Cough in the office can have many causes such as – an allergy to carpets, exposure to the air-conditioning, stuffy atmosphere, etc. You have not specified any of the above. Cough for 3 months continuously also may require proper investigations such as a routine blood test to check for Eosinophilia or a high ESR and a straight X-Ray of the chest. Please take Hepar Sulph 200 twice daily for 2 weeks. If there is no relief please consult a doctor regarding investigations.
2. I am 36.I have been suffering from ulcerative colitis for the past four years. My house physician prescribed a few medicines which didn't help at all. What shall I do?
Ans- If you have been suffering from Ulcerative Colitis for 4 years, your house physician is hardly likely to take it so casually as to "prescribe a few medicines …."! There is a definite well tested maintenance therapy for your problem in allopathy which includes drugs such as Salazopyrine and Mesacol etc. If you are on this drug we will need to continue them and tapered them off slowly. For a start, please take Carbolic Acid 6X in liquid form, 2 drops a dose, four times a day. Please consult a local Homeopath, as this disease requires proper monitoring.
3. I am 55 and have Cirrhosis of the liver. Last month I vomitted a few drops of blood. I have also been passing black stool. Please advise.
Ans- Cirrhosis of the liver is definitely not a disease to be treated casually on a newspaper column!! This is a serious condition and requires proper consultation and a good Homeopath on your locality. Bleeding in cirrhosis of liver can result from ruptured esophageal varices or hemorrhoids. In your case the bleeding is from the upper part of the GI tract, my bet would be that is from esophageal varices. You may temporarily take Ferrum Phos 3X and Arsenicum Album 3 – one dose every three hours alternately during the bleeds but please do not treat the matter lightly and get proper medical attention!
4 .I am a 45-yr-old man. Last yaer an endoscopy of my food pipe revealed an erosion in the lower stomach. Following a doctor's advise I took some alopathic drugs. I still have a burning sensation in the food pipe. What's wrong? Please advise.
Ans- Gastric erosions are very well treated by Homeopathy. Please improve meal times ie. take regular meals and take normal diet with spice and chillies and no the bland boiled diet that doctors often advise. To repair the erosions and improve the burning sensation in your food pipe please take - Arsenicum Album 3, twice daily and Lycopodium Clavat 200, every alternate morning. Arsenic Album 3 may be repeated incase there is burning as an SOS medicine too. Please take for 3 months for complete recovery.
5. I am a 30-yr-old man. In the last six months, I have lost three kilos. I have also lost appetite. No medicine has worked. Can you suggest a way out?
Ans- Loss of weight can be due to many factors and you have not been very detailed about what the possible cause can be. For a start, you should consult your local doctor who will probably suggest some investigation to determine the cause of your weight loss and loss of appetite. Just off hand, you may take Chelidonium Majus 6x, twice daily which will help with improving your appetite thus stem your weight loss. Though this will help, knowing the cause will help more to cure you in the long run.
6. I am 64, suffering from varicose veins and ulcers since 1980. It was operated in May 1992. Steroid cream had no tangible effect. It cured for the time being, please advise me.
Ans- Varicose veins and resultant avascular ulcers that result from it are a very difficult problem to handle with any school of medicine. The ulcers resulting from a lack of circulation in the tissues are often very painful and have a crippling effect on the unfortunate patient. People who have standing jobs – such as teachers or homemakers (standing and cooking) are at risk from this condition. Conventional medicine has hardly any effect on these cases and surgery is usually recommended, which is often of little use. Homeopathically, depending on the severity of the case, we prescribe the following, which will be of benefit to you – Hamamelis Verginica 200 + Arnica Montana 3 taken together twice daily, Hypericum 200 + Arsenicum Album 200 taken together twice daily. Please try for three months and see.
7. I suffered cerebral attack in 2003 and is now recuperating. My left side was Affected. Now I have recovered a little bit. Is there any homeo medicine you can suggest which can help me totally recover me from left side?
Ans- Cerebral attacks or Cerebro – Vascular Accidents (CVA) are usually a result of either a hemorrhage, embolism of infarction in the arteries of the brain. An accident in the left brain usually causes a paresis in the right side of the body and vice versa. Maximum improvement usually takes place within a month of the episode and all improvement usually stops within a year. The residual paresis usually persists and no further recovery takes place after that. Homeopathically, we have treated many cases of this condition after the one year period with varying degrees of further recovery. Please try the following – Rhus Tox 30 + Arnica Montana 3 taken together twice daily, Lachesis 200 every alternate morning for a period of three months and report to your local homeopath.
8. I am a 38-year-old woman. I don't have severe dandruff, but I am still turning Bald. What could be the problem? Please suggest me some remedy.
Ans- Causes of baldness are many and most often it has nothing to do with dandruff. Baldness mostly is caused by a disturbance in the hormonal patterns of the body. Different patterns of baldness result from disbalance in different hormones and addressing the imbalance can often help the problem. Homeopathically, we have some good medicines for hair loss – please try Ustilago 200 once daily and Sepia 200 twice a week.
9. I am a 60-year-old man. After drinking water, I feel such a strong urge to urinate That I cannot hold the bladder. X-ray of the abdomen revealed nothing. My doctor says the prostrate gland is fine, but there is a problem in the urinary tube. Is there any homeo remedy?
Ans- Urge incontinence of urine can happen due to a myriad of reasons but are more common due to a weakness / dysfunction in the sphincter (valve) of the urinary bladder. This causes the valve to "give way" as soon as the bladder fills to a certain level. Other reasons for the incontinence of urine include an irritability of the bladder wall muscles. Anyway please try the following. Medorrhinum 200 alternate morning, Cantharis 200 twice daily and observe.
10. My father 56 has been suffering from a persistent low-back pain for the last two Months. X-rays and ultra-sonogram have revealed no anomaly. But no medicine has been effective. My father has no other health problem. Could you suggest a remedy?
Ans- Diagnosis of the cause of low back pains are often elusive and due to this their treatment by allopathic medicines is usually done in a general way by the use of painkillers etc. Homeopathically, we have good medicines to help and the following will surely benefit him. Please give - Symphytum 200 twice daily, Hypericum 200 twice daily and Arnica Montana 1000 ( 2 drops in water) once in a week for month and see.


Adam Fields Wolf, 57 years old, died at home on April 2nd, surrounded by his loving family, after a courageous two-year battle with cancer.
Adam lived an extraordinary life filled with love, laughter, family and great friendships.
Born in Trumbull, CT, to Karen and George Wolf, Adam graduated from Trumbull High School in 1979 and then from Ohio Wesleyan University in 1983 with a BA in Communications followed by a Master's Degree in Corporate Political Communications from Fairfield University.
Adam married Pamela Wolf (nee Meyers) in 1990 and was her best friend and beloved husband for over 29 years. He was the incredibly loving and proud father of Haley Justine Wolf (25) and Morgan Kelsey Wolf (22), who were his greatest joys.
Adam was an amazing teacher to his daughters, showing them by example the importance of kindness, empathy and hard work. He was a man of true optimism, always rooting against all odds for his beloved NY Mets and NY Giants. Adam truly had the gift of gab, as evidenced by his wonderfully long and successful career in media sales. Most recently, Adam worked at NBC as a Director of New Business Advertising.
Adam's life was incredibly well-lived and well-loved. He approached each day with an excitement and energy that was as rare as it was infectious.
In addition to his loving wife and proud children, Adam is survived by his parents and his caring and supportive brother Daniel. Adam also will be deeply missed by cherished nephews, nieces, in-laws and countless friends. His loss is immeasurable to so many.
Services for Adam will be held at 12 p.m. on Friday April 5th at Congregation B'nai Israel, 2710 Park Avenue, Bridgeport, CT, 06604. In lieu of flowers the family asks for donations to be made in Adam's name to Dr. Jeffrey Bruce, Columbia Presbyterian Hospital, 710 168th St, Rm 434, New York, NY, 10032. Please make checks payable to Trustees of Columbia University and write Dr. Bruce Research in the memo portion of your check.


Rice, the lifeblood of so many nations' cuisines, is perhaps the most ubiquitous food in the world. In Asia, where an estimated 90 percent of all rice is consumed, the pillowy grains are part of almost every meal. In the Caribbean, where the starch is often mixed with beans, it's a staple too. Even here in the United States, where people eat a comparatively modest amount of rice, plenty is still consumed.
Rice is popular because it's malleable—it pairs well with a lot of different kinds of food—and it's relatively cheap. But like other starch-heavy foods, it has one central flaw: it isn't that good for you. White rice consumption, in particular, has been linked to a higher risk of diabetes. A cup of the cooked grain carries with it roughly 200 calories, most of which comes in the form of starch, which turns into sugar, and often thereafter body fat.
But what if there were a simple way to tweak rice ever so slightly to make it much healthier?
An undergraduate student at the College of Chemical Sciences in Sri Lanka and his mentor have been tinkering with a new way to cook rice that can reduce its calories by as much as 50 percent and even offer a few other added health benefits. The ingenious method, which at its core is just a simple manipulation of chemistry, involves only a couple easy steps in practice.
To understand what's going on, you need to understand a bit of food chemistry.
Not all starches, as it happens, are created equal. Some, known as digestible starches, take only a little time to digest, are quickly turned into glucose, and then later glycogen. Excess glycogen ends up adding to the size of our guts if we don't expend enough energy to burn it off. Other starches, meanwhile, called resistant starches, take a long time for the body to process, aren't converted into glucose or glycogen because we lack the ability to digest them, and add up to fewer calories.
A growing body of research, however, has shown that it might be possible to change the types of starches found in foods by modifying how they are prepared. At the very least, we know that there are observable changes when certain foods are cooked different ways.
Potatoes, for instance, go from having the right kind of starch to the less healthful kind when they are cooked or mashed (sigh, I know). The process of heating and cooling certain vegetables, like peas and sweet potatoes, can also alter the amount of resistant (see: good) starches, according to a 2009 study. And rice, depending on the method of preparation, undergoes observable chemical changes. Most notably, fried rice and pilaf style rice have a greater proportion of resistant starch than the most commonly eaten type, steamed rice, as strange as that might seem.
Understanding this, James and Thavarajva tested eight different recipes on 38 different kinds of rice found in Sri Lanka. What they found is that by adding a lipid (coconut oil in this case, because it's widely used in Sri Lanka) ahead of cooking the rice, and then cooling the rice immediately after it was done, they were able to drastically change its composition—and for the better.
So far they have only measured the chemical outcome of the most effective cooking method for the least healthful of the 38 varieties. But that variety still produced a 10 to 12 percent reduction in calories. "With the better kind, we expect to reduce the calories by as much as 50 to 60 percent," said James.
The prospect of lower calorie rice is a big deal. Obesity rates are rising around the world, particularly in the developing world, where people rely more heavily on cheaper food staples. China and India, which are already seeing rising obesity problems, are huge consumers of rice. Rice, of course, is not the sole cause of weight gain. But reducing the amount of calories in a cup of rice by even as little as 10 percent could have an enormous impact for future generations.
The researchers still have to test the remaining varieties of rice, including Suduru Samba, which they believe will produce the largest calorie reduction. They also plan to experiment with oils other than coconut oil, like sunflower oil.
A world where commercially sold rice comes pre-cooked and with much fewer calories might not be that far off. People should already be able to replicate the process at home, although James warns the results might vary depending on the type of rice used. And there's good reason to believe the chemistry could be applied to many other popular but less-than-healthy foods.
This entry was posted in Rice and tagged 38 different kinds of rice, diabetes, Dr. Pushparajah Thavarajah, food chemistry, Rice, Sudhair James. Bookmark the permalink.


Bollywood Actors Sushant Singh Rajput and Sara Ali Khan promoted their film Kedarnath on the sets of Sony TV's Indian Idol 10 reality show. They have had great time with the Judges, Hosts and Top 7 Contestants on the Show. Also there is going to be an elimination during the 17th November 2018 and 18th November 2018. See Who are eliminated from Indian Idol 10 reality show.
After the Eviction of one more contestant from the show we'll have the Top 6 Singer Contestants Finalists will remain into the competition. Vote and Support your Favorite Singer in Top 7 list who are Renu Nagar, Nilanjana Ray, Salman Ali, Nitin Kumar, Ankush Bhardwaj, Vibhor and Kunal Pandit are the Top 7 Singer Contestants in a Show.
The Show Guests Sushant Singh Rajput and Sara Ali Khan promoted their film Kedarnath on the sets of Indian Idol 10 reality show.
Keep Watching Indian Idol 10 every Saturday-Sunday 8 PM on Sony TV, Vote and Support your favorite Artist in a Show.
we have better things to do than watch this show where there is injustice done. Soumya does not have any supporters in the show therefore he has be underrated. Javed Ali has a problem with him. i am sure he did not give Soumya full points. Very unfair!


Snapchat is more fun in my MADMIA Snap Chat Socks.
MADMIA Snap Chat Socks are a mix and match pair of pink and yellow with prints of social media and text acronyms and characters. Complete with shoelace print and shoe lace topper of yellow and pink.
Snapchat Socks are suitable for Kids and Teenegers age 6 and up. These Socks can fit adults but only up to shoe size EU 41 equivalent to womens size 9.


Look through the listings of Member singles here at Over 40 Singles Club that are associated with Water polo. Meeting other singles who have similar interests is a great way to come up with ideas to do on a first date. Create a 100% Free Profile to Find your perfect match!


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Sathya Life Coach
Life Coach | HR Professional
Benefits of Coaching
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Leave a Comment / Uncategorized / By SathLifeAdmin
That, coaching works like magic and enables one to delve deeper and discover blind spots/limiting beliefs is a known fact. But the process of witnessing this magic unfold in ways one might never expect during the course of a coaching session is an overwhelming experience. I strongly believe in the words of Paulo Coelho that, "When you want something, all the universe conspires in helping you to achieve it". This quote truly resonates with me every time i partner with a client on a journey of growth/progress.
One of my clients had come forward to speak about her multifarious roles spread across work and home. She also spoke about how she was receiving no credit/acknowledgement from any side for all her efforts and how she was feeling overwhelmed. Through a visualization exercise, she was able to take me through a typical scenario of what exactly transpires and how it leads to this feeling of "lack of acknowledgement". No matter how many times she said that, she didn't want to care about seeking external validation, every-time she ended by saying that there was no credit/acknowledgement. After a brief silence, i asked her "what was the common trend emerging across all these situations?". She said that she may say that external validation is not required but then she craves for a little bit of appreciation. She also noticed that, no one was expecting her to do anything out of the way, but it was she, who wanted to ensure everyone and everything was taken care of. When further asked, "If you were to ask your higher self, about the roles that you play across work and life, what would be the response?". She thought briefly for a moment and said that, her higher self would laud her for giving her best in each situation and doing all that she can to the best of her knowledge. But also stated that, in her quest for external validation, she had ignored her own inner self which would give her a better view of what she wanted to know. This was such a powerful realization not just for her but also for me. Sometimes all the answers that you ever wanted to hear, lie within you! In fact, all that you want to ever achieve, becomes absolutely possible, when you look within just to understand that you can simply tap into the resources and capabilities you have been endowed with.
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Honus Honus | portrait by Mustafa Shaheen | courtesy of the artist
Listen to a new song from Honus Honus called "Oh No!"
With his debut solo LP set for release next month, Honus Honus has shared a new track called "Oh No!" via Consequence of Sound. Also known as the frontman for Philly-based Man Man (and in some circles, Ryan Kattner), Honus keeps the colorfully weird musical sensibilities we love about his band alive on Use Your Delusion.
As Kattner tells CoS, the solo LP came about during a hiatus period for Man Man. The band had released 2013's On Oni Pond, toured all over and needed a well-earned bit of downtime. But Kattner doesn't really slow down like that, so here we are three years later with a complex, fully-realized psych-pop record chock full of collaborations from his newly adopted home of Los Angeles.
About "Oh No!," Kattner writes:
"[Producer King Cyrus King] wasn't a fan of the song when I initially brought it to the table so it took a bit of convincing to get him onboard. It truly was a situation where when he first heard me demoing it out he went, 'Oh no, not this song!' The name was sort of born out of that moment even though the lyrics in the bridge already hinted at a title. Also, 'Ono' in this pronunciation is Hawaiian for delicious. Food for thought.
Going into this making this record I wanted to play around with different synth sounds, experiment with brighter, maybe even 'cheesier' tones that would draw a sharp contrast to some of the non-breezy lyrical content. I've always been a fan of juxtaposing opposing vibes and I love how the tune has a bouncy, summer feel but if you actually listen to what I'm singing, the sentiment is rather melancholy since it's a song dealing with a breakup and post-breakup healing. But…if you're someone who just hears the music and could care less about what the song is about, it's a feel-good jammer and sounds even better when you pump up the volume! True story."
Catch Honus Honus at Boot & Saddle on November 15th; tickets and information for the 21+ show can be found here. Want more? Read our interview with him about the new record here.


With Legal Steroids you can embark on a journey that will change the way you approach muscle building and fat loss forever.If you're not the most genetically gifted person, these Legal Steroids will accelerate muscle growth and strength faster than any other bodybuilding supplement or Legal Steroid on the market. If you are one the few genetic elites, anabolic supplements Legal Steroids will take advantage of your genetics and accelerate your muscle growth much faster than "normal" bodybuilding supplement aids. Male or female – it doesn't matter. Simply put, for building muscle; Legal Steroids is the safest and most effective choice.
Whether you're looking to pack on a massive 50 pounds of muscle or a trim and tight 5 pounds,then Legal Steroids is the most effective way to accomplish this. From the highest level competitor to the most casual lifter, Legal Steroids is the most sensible approach to reaching your goals in the shortest amount of time.
Legal steroids, technically known anabolic supplements or prohormones, are muscle enhancers that structurally related to the cyclicsteroid ring system. Similar effects to testosterone in the body. They increase protein within cells, especially in skeletal muscles. Anabolic steroids also androgenic – like and virilizing properties. Including the development and maintenance of masculine characteristics like muscle growth and fat loss. Also testicles (primary sexual characteristics) and body hair (secondary sexual characteristics). The word anabolic comes from the Greek ἀναβολή anabole, "that which is thrown up, mound", and the word androgenic from the Greek ἀνδρός andros, "of a man" + -γενής -genes, "born".
By technical definition, ANABOLIC refers to protein synthesis which aids the body in muscle building, fat loss, and the development of strength.
Anabolism , or the The Anabolic process (from Greek: ἁνά, "upward" and βάλλειν, "to throw") is the set of metabolic pathways that construct molecules from smaller units. These reactions require energy, known also as an endergonic process. One way of categorizing metabolic processes, whether at the cellular, organ or organism level, is as "anabolic" or as "catabolic", which is the opposite and thus the separation of a macromolecule. Anabolism is powered by catabolism, where large molecules are broken down into smaller parts and then used up in cellular respiration. Many anabolic processes are powered by the hydrolysis of adenosine triphosphate (ATP).
Anabolic processes tend toward "building up" organs and muscle tissue. These processes produce muscle growth and differentiation of cells and increase in body size, a process that involves synthesis of complex molecules. Examples of anabolic processes include the growth and mineralization of bone and increases in muscle mass. Endocrinologists have traditionally classified hormones as anabolic or catabolic, depending on which part of metabolism they stimulate. The classic anabolic hormones are the anabolic steroids, which stimulate protein synthesis, muscle growth, and insulin. The balance between anabolism and catabolism is also regulated by circadian rhythms, with processes such as glucose metabolism fluctuating to match an animal's normal periods of activity throughout the day.


Please submit the Application Form by 20 March 2019. Results of the application procedure will be announced by e-mail by 1 April 2019 at the latest.
14. Do you have any food allergies or dietary requirements?
15. Please write in a few sentences why you wish to participate in the staff week.


Professor Marc Cowling has a PhD in Business Economics from Warwick Business School and an MSc Economics from London University. Before his appointment at Brighton he was Professor and Head of the Department of Management Studies at Exeter Business School. Prior to that, he held the posts of Chief Economist at the Institute for Employment Studies and The Work Foundation. He has also held positions at Warwick Business School, Birmingham Business School (where he was Director of the Research Centre for Industrial Strategy) and London Business School (where he was Deputy Principal Investigator of the Global Entrepreneurship Monitor). He is currently ranked in the top 12% of economists in the world by citations (H-index) according to Research Publications in Economics (REPEC, May 6th 2014) and in entrepreneurship he was ranked 23rd in the world over the period 1995-2006 according to "Rankings of the Top Entrepreneurship Researchers and Affiliations.


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Q: what is in background of html submit form? I'm trying to figure out what really happens when html submit form button is clicked.
I suppose it generates some kind of http request (similar to ajax get or post call) which has data in http body and is sent to address specified in action field.
1) Am I right?
2) I've seen many ways of processing forms with PHP or ASP on server side. Can I process it with Java REST Application using e.g. Jersey? Is submit form capable of hitting REST if I put right URL in action field?
Thank You.

A: By submitting the form in HTML you basically tell the browser to generate a normal HTTP request, usually POST or GET, for an URL defined in  tag with form fields attached according to the specified method either appended to the URL or included in the request data.
There is nothing really special or different from a "normal" HTTP request, in fact you can manually "submit a form" by appending form keys and values to the URL in your browser and navigating to it in case of GET method.
Summarizing:
1) Yes, you are right.
2) From what I've just read (never used REST personally) a REST application is implemented by a servlet mechanism and uses HTTP protocol, so it should be possible to write a REST application for processing HTML forms if the form points to this application's URL.

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