Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, while the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C-terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.

*e-mail: douglasm@illinois.edu, phone: 1-217-333-1345, fax: 1-217-333-0508 *e-mail: snair@uiuc.edu, phone: 1-217-333-0641, fax: 1-217-244-5858     Table 1. Oligonucleotide primers used in this study. Mutations are listed in parentheses, asterisks denote stop codons, and endonuclease cut sites are listed when appropriate. The *430A mutation indicates that the BalhD stop codon was mutated to alanine. This resulted in an additional 12 residues to the C-terminus of BalhD (-A 4 LEH 6 ). F, forward primer; R, reverse primer.  Table 3. List of peptides used in this study. Residues known to be cyclized in vitro or in vivo are colored orange. In McbA, the orange underlined serine (CSN) is the site of the ninth heterocycle that is installed in vitro and found as a minor microcin B17 species 1 . A caret and an asterisk denote putative and known leader peptide cleavage sites, respectively. FP-BalhA1-LP is a synthetically prepared reagent designed for fluorescent polarization (FP) studies. The reagent contains a fluorescein label (installed via the amine reactive isothiocyanate, FITC) linked to an aminohexanoic-glycine spacer, which in turn is linked to the predicted leader peptide (LP) of BalhA1.

Supplementary Figure 3. Ec-YcaO hydrolyzes ATP to AMP and pyrophosphate. (a)
The ATPase activity of Ec-YcaO was screened using the purine nucleoside phosphorylase (PNP)-coupled assay 8 with and without 1 unit of pyrophosphatase (PPase). The addition of PPase increased the rate of chromophore production in the assay by 5-fold after the signal amplification achieved during PP i cleavage is taken into account. These data indicate that ATP is preferentially hydrolyzed to AMP and pyrophosphate by Ec-YcaO in vitro. This result is corroborated by the observation that co-crystallization of Ec-YcaO with ATP yielded an AMP-bound structure ( Fig. 2A). Error bars represent the standard deviation from the mean (n=3). (b) An ATP kinetic curve was obtained for Ec-YcaO using the PPase-supplemented PNP assay. Error bars represent the standard deviation from the mean (n=3). Regression analyses to obtain Michaelis-Menten kinetic parameters were carried out in IGOR Pro version 6.12 (Wavemetrics). The error on the kinetic parameters represents the standard deviation from the curve fitting. Residues mutated in BalhD for this study are denoted by black triangles. A summary of the BalhD mutants made, along with their predicted roles in ATP recognition is provided.  Figure 5. Cytoscape sequence similarity network for the YcaO superfamily. A similarity network for non-redundant YcaO sequences is displayed. Each node represents a unique YcaO, while lines between nodes exist if the proteins bear significant similarity (in this case, a BLAST expectation value lower than 10 -80 ) . At this e-value, YcaO proteins from characterized TOMM biosynthetic clusters primarily form isofunctional clusters. TOMM YcaOs are defined as having a bioinformatically identifiable C protein within 10 kb of the YcaO protein. Stand-alone TOMM YcaOs are proteins found in a characterized TOMM biosynthetic clusters that lack a C protein (e.g. bottromycin and trifolitoxin). It is noteworthy that many non-TOMM YcaOs appear to exist in RiPP biosynthetic gene clusters, indicating that additional stand-alone YcaO cyclodehydratases may exist. Nodes are colored according to the legend and the clusters containing Ec-YcaO and BalhD are indicated.

Supplementary Figure 6. Diversity oriented Maximum likelihood tree for the YcaO family.
A diversity-maximized tree for the YcaO superfamily is displayed. Red asterisks denote YcaOs associated clusters of functionally or structurally characterized natural products, while the proteins used in this study are circled and colored blue. The members of this tree were used to generate WebLogo 9 frequency plots for the conservation of the ATP-binding residues in TOMM, non-TOMM, and TfuA-associated non-TOMM subclasses of the YcaO superfamily (Fig. 3A). ). An alignment of diverse non-TOMM E1 proteins was generated and the ATP-and Mg 2+ -binding residues identified in these crystal structures were highlighted in orange. The two CxxC motifs responsible for coordination of the structural Zn 2+ found in all characterized E1 ubiquitin-activating family members are highlighted in blue [15][16][17] . To improve the alignments, the highly variable N-terminal "peptide clamp" domain found in MccB homologs was manually removed. A sequence identity matrix is provided to demonstrate the level of divergence between members of the family. In spite of the divergence, the alignments demonstrate that the ATPbinding pocket is remarkably conserved among these non-TOMM E1 members. (b) Alignments of TOMM C proteins associated with cyanobactin biosynthesis to E. coli MoeB, ThiF and MccB are displayed. The highly variable N-terminal residues of each C protein were removed as above.
Additionally, the YcaO domain of each CD fusion was removed. Unlike the non-TOMM E1 superfamily members, the TOMM C proteins show little conservation in the canonical E1-like ATP-binding site. This suggests that the ability to bind/utilize ATP has been lost in the TOMM C proteins. While non-TOMM E1 sequences are identified by GI number and the subclass of the E1 superfamily they belong to, TOMM E1 sequences are identified by they natural product that they produce.

Supplementary Figure 8. The ATP-binding pocket is conserved in characterized TOMM YcaOs.
All of the YcaO sequences from the indicated TOMM subclasses were aligned with Clustal Omega 10 and the multiple sequence alignments were used to generate sequence logos (WebLogos) 9

Supplementary Figure 10. Mutations to the BalhD ATP-binding site affect heterocycle formation.
A MALDI-TOF MS spectral overlay for BalhA1 treated with BalhC and either wild-type (WT) or mutant BalhD is displayed. Many of the mutations to the ATP-binding residues, which are listed on the right, decreased cyclodehydratase activity. The level of processing is summarized in Table 1. †, laserinduced deamination. A loss of 90 Da indicates the formation of 5 azolines, a full in vitro processed BalhA1 substrate 11 .
Supplementary Figure 11. Mutations to the BalhD ATP-binding site affect ATP hydrolysis. The rate of ATP hydrolysis was measured with BalhC and either wild-type (WT) or mutant BalhD using the PNP assay. Error represents the standard deviation from the mean (n ≥ 3). Mutations showing the slowest rate of ATP hydrolysis also installed fewer heterocycles on BalhA1 in an endpoint assay (Supplementary Fig. 10).
WT S72A E76A E79A Q186A N190A E194A E197A R198A K281A E286A R80A  Figure 12. Mutations to the ATP-binding site that decrease cyclodehydratase activity also affect BalhD-only activity. MALDI-TOF MS spectra for BalhA1 treated with wild-type (WT) or mutant BalhD is displayed. Apart from BalhD S72A, all of the mutants displayed a decreased level of D-only processing consistent with the rate of ATP hydrolysis measured in the presence of the C protein (Supplementary Fig. 11). The level of processing is summarized in Table 1. †, laser-induced deamination.
Supplementary Figure 13. The K M for ATP does not depend on the concentration of BalhA1. Due to solubility limitations, saturating concentrations of BalhA1 were not obtainable for all of the BalhD mutants. To determine if the K M for ATP changed at non-saturating concentrations of BalhA1, an ATP kinetic curve was carried out at the K M for BalhA1 (15 µM). The resultant K M is within error of the previously reported K M for ATP, 240 ± 20 µM, obtained with a saturating concentration of BalhA1 18 . Error on the Michaelis-Menten parameters represents the standard deviation from the regression analysis.  a dimer (2). When the proteins were mixed, the intensity of the BalhC dimer peak decreased and a new peak corresponding to the BalhC/D complex appeared (1). Absorbance values were normalized to the peak at ~3.5 mL. (b) To better quantify the BalhC binding affinity of the mutant BalhD proteins, ATP hydrolysis rates were monitored for wild-type cyclodehydratase reactions with the addition of variable concentrations of inactive BalhD mutants. As the reactions were carried out with 1 µM wild-type BalhD, mutants with an unperturbed BalhC interaction would display IC 50 values of approximately 1 µM. Error bars represent the standard deviation from the mean (n ≥ 3). Regression analysis to determine the IC 50 values for each inhibition curve was determined in IGOR Pro version 6.12 (Wavemetrics). The error on the IC 50 values represents the standard deviation from the curve fitting.

(a)
A MALDI-TOF MS spectral overlay for BalhA1 treated with BalhC and either wild-type (WT) or mutant BalhD is displayed. The level of processing is summarized in Table 2. (b) The rate of ATP hydrolysis was measured with BalhC and either wild-type (WT) or mutant BalhD using the PNP assay. Error represents the standard deviation from the mean (n ≥ 3). *, stop codons; †, laser-induced deamination; ∆2 AA, BalhD (∆A418, K419).

Supplementary Figure 18. Mutations that relocate the C-terminus of BalhD prevent ATPase potentiation by BalhC.
The background ATP hydrolysis rate of BalhD C-terminus mutants lacking the ability to modify BalhA1 was measured with and without BalhC. Although the mutants displayed a higher rate of ATP hydrolysis than wild-type BalhD, none of the mutants could be potentiated by the addition of BalhC (P > 0.1). Error bars represent the standard deviation to the mean (n ≥ 3). For reactions with BalhC, the horizontal lines indicate the expected rate of ATP hydrolysis with the background ATPase rate of BalhC. *, stop codons; ∆2 AA, BalhD (∆A418, K419).  BalhD retained as a monomer (3), BalhC had a retention volume consistent with a dimer (2). When the proteins were mixed, the intensity of the BalhC dimer peak decreased and a new peak corresponding to the BalhC/D complex appeared (1). Absorbance values were normalized to the peak at ~3.5 mL. (b) ATP hydrolysis rates were monitored for wild-type cyclodehydratase reactions with the addition of variable concentrations of a C-terminal BalhD truncated protein. As the reactions were carried out with 1 µM wild-type BalhD, mutants with an unperturbed BalhC interaction would display IC 50 values of approximately 1 µM. *, stop codon. Error bars represent the standard deviation from the mean (n ≥ 3). Regression analysis to determine the IC 50 values for each inhibition curve was determined in IGOR Pro version 6.12 (Wavemetrics). The error on the IC 50 values represents the standard deviation from the curve fitting. . Stars indicate the location of the azole heterocycles. In the di-azole species, the second heterocycle is found at both Cys45 and Cys31. The MS/MS spectrum for the di-azole species is shown in (c). Cys45 is the penultimate residue cyclized by a wild-type Balh cyclodehydratase 11,13 . As such, these data indicate that the ring order of the complex has been perturbed in the P429* mutant. These data indicate that the sequence of the C-terminus of BalhD is critical for cyclodehydratase activity. The level of processing is summarized in Table 2. (b) The rate of ATP hydrolysis was measured with BalhC and either wild-type (WT) or mutant BalhD using the PNP assay. Error represents the standard deviation from the mean (n ≥ 3). These data provided further evidence that the C-terminal residues of BalhD are critical for cyclodehydratase activity. †, laser-induced deamination. The wild-type sequence for the C-terminal PxPxP is 425 PHPFP 429 .

Supplemental Figure 22. The sequence of the C-terminus of BalhD is important for binding BalhC. (a)
Size-exclusion chromatography was used to monitor the effect the PxPxP-tail mutations had on complex formation. Although BalhD retained as a monomer (3), BalhC had a retention volume consistent with a dimer (2). When the proteins were mixed, the intensity of the BalhC dimer peak decreased and a new peak corresponding to the BalhC/D complex appeared (1). Absorbance values were normalized to the peak at ~3.5 mL. (b) ATP hydrolysis rates were monitored for wild-type cyclodehydratase reactions with the addition of variable concentrations of a catalytically deficient BalhD mutant. As the reactions were carried out with 1 µM wild-type BalhD, mutants with an unperturbed BalhC interaction would display IC 50 values of approximately 1 µM. All of the BalhD mutants had a disrupted BalhC affinity. (c) As the competition assay is only compatible with BalhD mutations displaying almost no activity, the interaction between F428A and P429G could not be determined with this assay. For these active mutants, the interaction between BalhC and mutant BalhD was interrogated by measuring an apparent "K M " for BalhC. A BalhC "K M " was obtained by holding the concentration of mutant BalhD and BalhA1 constant and adding variable concentrations of BalhC. The rate of processing was monitored using the PNP-phosphate detection assay. For clarity, the initial processing rates were normalized to the rate obtained with a 1:1 ratio of BalhC and mutant BalhD. Together, these data indicated that the prolines in the PxPxP motif are more important for BalhC recognition than the intervening "x" residues and that residues closer to the active site (more C-terminal) are less important for BalhC binding. Error bars represent the standard deviation from the mean (n ≥ 3). Regression analysis to determine the IC 50 and K M values for each inhibition curve was determined in IGOR Pro version 6.12 (Wavemetrics). The error on the IC 50 and K M values represents the standard deviation from the curve fitting. Although BalhD P429G had an initial rate of substrate processing that is 1/3 of wild-type BalhD, the mutant produced primarily a tetra-ring species following a 16 h reaction. Based on the aberrant ring order observed in BalhD P429* reactions, we hypothesized that the inability to generate the penta-azole species could be due to perturbation of ring order. To locate the sites of heterocyclization, BalhA1 was treated with BcerB, BalhC, and BalhD P429G for 16 h at 25 ˚C. (a) A high-resolution, intact mass spectrum of the processed substrate is displayed. The error (ppm) associated with each measurement from the calculated m/z is shown. (b) MS/MS fragmentation of the 5, 4 and 3 ring species was performed and the resultant fragmentation maps are displayed. The b-and y-ions are colored based on the number of azoles heterocycles in the fragment (black = 0, orange = 1, purple = 2, blue = 3, pink = 4, green = 5). Stars indicate the location of the azoles. In the tri-azole species, the third heterocycle was found at both Cys45 and Cys34. The MS/MS spectrum for the tri-azole species is shown in (c). Based on the b-and y-ion intensities, both positions appeared to be cyclized to approximately the same extent. In wild-type reactions, the tri-azole species has rings at Cys31, Cys34, and Cys40 11,13 . The deviation from the wild-type processing order was not observed in the tetra-or penta-azole species. Although less severe than the dysregulation in cyclization order found with BalhD P429*-treated BalhA1 (Supplementary Fig. 20), the identification of partial processing at Cys45 in the tri-azole species suggested that the BalhC/D interaction was perturbed. Based on the data from this manuscript, a model for azoline formation is provided. The TOMM E1 homolog (C protein) allosterically activates the YcaO domain (D protein) and binds the leader peptide (LP). Leader peptide binding by the C protein places the core peptide in proximity of the YcaO active site to facilitate the cyclodehydration reaction.