Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain
Amyloid fibrils are protein deposits often associated with disease . These deposits show common structural and biochemical characteristics , including non-covalent self-complementary fibrils , resistance to proteolysis , insolubility in aqueous solution , binding of thioflavin dye , and generation of a cross-β X-ray diffraction pattern . The spine of amyloid fibrils , termed a 'steric zipper , ' is built by paired β-sheets with interdigitating side chains and serves as the scaffold for fibril extension . The inter-strand and inter-sheet distances give rise to a characteristic cross-β X-ray diffraction pattern of two perpendicular reflections at ~4 . 8 and ~10 Å ( Geddes et al . , 1968; Hobbs , 1973; Sipe and Cohen , 2000; Nelson et al . , 2005 ) . Formation of a steric zipper requires the presence of a discrete peptide segment with an amino acid sequence capable of forming a self-complimentary propagating structure . Such segments are commonly found as integral parts of globular proteins and trigger aggregation into amyloid fibrils only upon exposure to solvent through full or partial denaturation ( Nelson et al . , 2005 ) . Cases of systemic light chain amyloidosis ( AL ) , associated with multiple myeloma , were first described in the mid-19th century; however , the etiology of the disease was not clear . In 1848 , Dr . Bence-Jones discovered the disease-associated substance in urine , though it was only later identified as immunoglobulin light chains ( LC ) associated with amyloidosis ( Kyle , 2001; 2011; Dahlin and Dockerty , 1950; Glenner et al . , 1970; 1971; Glenner , 1973; Sipe et al . , 2014 ) . Systemic AL amyloidosis is characterized by overexpression of monoclonal LCs , which are able to form homo-dimers . Despite renal clearance of excess protein , the full-length LCs or their fragments still form amyloid fibril deposits in patients’ tissues , most frequently in the heart and kidneys , which result in organ failure ( Falk et al . , 1997; Buxbaum , 1986 ) . Although it is clear this deposition causes organ dysfunction , much remains to be discovered about the molecular process by which immunoglobulins assemble into amyloid fibrils . It is unknown which peptide segments form steric zippers and how to block their assembly into the amyloid spine . LCs are produced by plasma cells and their final amino acid sequence is determined by somatic recombination ( Sakano et al . , 1979; Marchalonis and Schluter , 1989 ) . LCs are classified by their amino acid sequence as either κ or λ . They consist of two domains , the variable ( VL ) and constant ( CL ) , which are connected by a joining segment ( J ) . In patients , amyloid fibrils are found to include either VLs or full-length LCs ( VL-J-CL ) , yet the ubiquitous presence of VLs indicates that this domain may be the minimal and essential unit for fibril assembly ( Falk et al . , 1997; Buxbaum , 1986; 1992; Olsen et al . , 1998; Lavatelli et al . , 2008; Vrana et al . , 2009; Bodi et al . , 2009 ) . The crystal structures of full-length LCs and structures of just their VLs both reveal homo-dimers , which resemble the light chain and heavy chain hetero-dimer of the antigen-binding fragment ( Fab ) of a full antibody . Fab is a hetero-dimer composed of both light and heavy chains , each with constant ( CL and CH ) and variable ( VL and VH ) immunoglobulin domains . LC homo-dimers , in which the VL domain is covalently linked to the CL domain , resemble the quaternary structure of the Fab hetero-dimer , and non-covalently linked dimers of only VLs structurally resemble the variable region of the Fab ( VL and VH ) ( Edmundson et al . , 1969; Colman et al . , 1977; Firca et al . , 1978 ) . The interfaces between the variable domains of physiological hetero-dimers and pathological homo-dimers are lined with apolar residues encompassing a hydrophobic cavity capable of accommodating small molecules ( Edmundson et al . , 1984; 1993 ) . Our work focuses on a λ patient-derived VL homo-dimer named Mcg whose structure and properties were detailed in pioneering studies by Edmundson and coworkers ( Edmundson et al . , 1969; 1984; 1993; Firca et al . , 1978 ) . The cavity between the two VL domains in Mcg is capable of binding a wide array of ligands , including synthetic organic molecules and peptides , which form contacts with the side chains of the dimer cavity . The cavity spans most of the 24 Å-long dimer interface; it is cylindrically shaped and can be considered to constitute three sites: A , B and C ( Edmundson et al . , 1984 ) . Based on the geometry of the dimer with some deviation from the original nomenclature by Edmundson et al . , we designate side chains of residues Y34 , Y93 , D97 and F99 on one side of the cylindrical cavity as the 'A-site , ' S36 , Y51 , E52 , S91 and F101 as the 'B-site , ' and Y38 , Q40 , V48 and Y89 as the 'C-site' ( Figure 1 ) . Some ligands bind the cavity in a sequential manner without a distinct preference for one of the sites . For example , 1-anilinonaphthalene-8-sulfonic acid binds in either the A or C-sites and can migrate between them , whereas fluorescein binds to both sites with partial occupancy . Other molecules , such as menadione , preferentially bind to only one of the sites ( Edmundson et al . , 1984 ) . Regardless of binding location , this demonstrates the dimer cavity is capable of accommodating various hydrophobic and aromatic ligands . 10 . 7554/eLife . 10935 . 003Figure 1 . Stereo image of the ligand-binding sites of the VL dimer . We designate the A-site in red ( residues Y34 , Y93 , D97 and F99 ) , B-site in yellow ( residues S36 , Y51 , E52 , S91 and F101 ) , and C-site in green ( residues Y38 , Q40 , V48 and Y89 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 003 VLs exist in equilibrium between homo-dimers and amyloid-prone monomers . Experiments conducted in denaturing conditions indicate that reducing the stability of the monomeric state promotes amyloid fibril formation , and mutations that induce dimer disassociation or promote monomer unfolding increase the propensity to form amyloid fibrils ( Bernier and Putnam , 1963; Kishida et al . , 1975; Qin et al . , 2007; Wetzel , 1994; Hurle et al . , 1994; Brumshtein et al . , 2014; Baden et al . , 2008 ) . Likewise , mutations that stabilize the structure of VLs or covalently fix VL dimers inhibit formation of amyloid fibrils . These results indicate that formation of amyloid fibrils involves two steps: VL dimer disassociation into monomers followed by partial or full unfolding . The mechanism of amyloid formation also suggests that shifting the equilibrium away from the amyloid-prone monomer by stabilizing the dimer would hinder formation of amyloid fibrils ( Figure 2 ) ( Bulawa et al . , 2012; Bellotti et al . , 2000 ) . 10 . 7554/eLife . 10935 . 004Figure 2 . Proposed mechanism for using ligands to hinder the aggregation of immunoglobulin VL s into amyloid fibrils . VL s are in equilibrium between dimers and monomers in solution . Ligands may be used to stabilize the VL dimer and therefore shift the equilibrium away from amyloid-prone monomers . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 004 The monomer-dimer equilibrium of VLs suggests that systemic AL amyloidosis may be mitigated by binding ligands to the cavity at the VL dimer interface ( Figure 2 ) . This approach proved effective for transthyretin-related amyloidosis , another type of systemic amyloidosis for which stabilizing the quaternary state led to the development of therapeutics ( Miroy et al . , 1996 ) . Upon transthyretin tetramer disassociation into amyloid-prone monomers , it forms amyloid fibrils in an acidic environment . The binding of thyroxine inhibits disassociation and subsequent amyloid formation ( Baures et al . , 1998 ) . Following the same principle , a modified ligand with a disassociation constant in the nano-molar range prevents transthyretin from forming amyloid fibrils and is effective in vivo . Here we apply structural and biochemical methods to investigate ligands that hinder amyloid formation by stabilizing the VL homo-dimer . We identify ligands that may serve as prototypes for therapies for treating LC amyloidosis and our results are consistent with a mechanism for amyloidosis that proceeds via dimer disassociation to amyloid-prone monomers ( Qin et al . , 2007; Brumshtein et al . , 2014 ) .
Identification of ligands that inhibit formation of amyloid fibrils . Based on the previous work of Edmundson et al . , we screened a panel of 27 hydrophobic and aromatic ligands for an inhibitory effect on the formation of amyloid fibrils ( Figure 3 ) ( Edmundson et al . , 1976; 1984; 1993 ) . Such ligands may stabilize VL dimers and decrease disassociation into amyloid-prone monomers . Varying concentrations of ligands up to 1 mM were used to evaluate the effect of complexes on formation of amyloid fibrils . Formation of amyloid fibrils was monitored using both ThT fluorescence assays and EM micrographs . Molecules that showed an inhibitory effect with concentrations under 1 mM were 8-anilino-1-naphthalene sulfonic acid [5] , methylene blue [10] , Chicago Sky Blue 6B [14] and Phenol Red [20] ( Figure 3B ) . 10 . 7554/eLife . 10935 . 005Figure 3 . Panel of ligands analyzed with ThT assays and EM for their ability to inhibit VL amyloid formation . ( A ) Preliminary molecules screened . 1 . 1 , 2-naphthoquinone . 2 . 1 , 4-naphthoquinone . 3 . 5-hydroxy-1 , 4-naphthoquinone . 4 . quinone . 5 . 8-anilinonaphthalene-1-sulfonic acid . 6 . methoxy-2-naphthyl-propionic acid . 7 . dopamine . 8 . L-phenylalanine . 9 . L-tryptophan . 10 . methylene blue . 11 . eosin Y . 12 . rhodamine 6G . 13 . Basic Blue 12 . 14 . Chicago Sky Blue 6B . 15 . 8-aminonaphthalene-1 , 3 , 6-trisulfonic acid . 16 . Basic Blue 41 . 17 . dimenhydrinate . 18 . stictic acid . 19 . 6-carboxyfluorescein . 20 . phenol red . 21 . R- ( - ) -apomorphine . 22 . sulforhodamine 101 . 23 . fluorescein methyleneiminodiacetic acid . 24 . rhodamine B . 25 . tetracycline . 26 . doxycycline . 27 . ( − ) -epigallocatechin gallate . ( B ) Molecules showing an inhibitory effect on the formation of amyloid fibrils during the preliminary screen . ( C ) Molecules identified in a DrugBank search using the most effective candidates from the preliminary screen and a similarity threshold of 0 . 3 . Search using phenol red: 28 . diethylstilbestrol ( 0 . 4 ) , 29 . sulindac ( 0 . 4 ) , 30 . tamoxifen ( 0 . 4 ) , 31 . acitretin ( 0 . 4 ) . Search using Chicago Sky Blue 6B: 32 . suramin ( 0 . 4 ) , 33 . Bumetanide ( 0 . 3 ) , 34 . sulfasalazine ( 0 . 3 ) , 35 . sulfadoxine ( 0 . 3 ) , 36 . amsacrine ( 0 . 3 ) , 37 . methotrimeprazine ( 0 . 3 ) . Search using methylene blue: 38 . clofazimine ( 0 . 3 ) , 39 . primaquine ( 0 . 3 ) , 40 . imiquimod ( 0 . 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 005 Methylene blue , Chicago Sky Blue 6B , and Phenol Red ( 10 , 14 and 20 ) were used to search DrugBank for approved biomedical compounds resembling their chemical structures and properties with a similarity threshold value of 0 . 3 ( Law et al . , 2014 ) . The search identified 13 compounds that were tested for an amyloid inhibitory effect by means of ThT assays and EM analysis ( Figure 3C ) . Of these 13 molecules from DrugBank , only sulfasalazine [34] showed an inhibitory effect on formation of amyloid fibrils . Among all the molecules tested , including the ligands initially screened and the subsequent approved biomedical compounds , the two that show the most potent effect are methylene blue [10] and sulfasalazine [34]; both hinder amyloid fibril formation at effective concentrations of 0 . 1 and 0 . 5 mM respectively ( Figure 4 ) . 10 . 7554/eLife . 10935 . 006Figure 4 . Thioflavin T fibril formation assays and electron micrographs of Mcg VLs in the presence of various concentrations of methylene blue and sulfasalazine . Averaged fluorescence readings are based on three repeated ThT assays . Error bars are not shown for clarity . The EM micrographs have a scale bar of 0 . 5 μm . ( A ) Mcg alone . ( B ) Mcg with various concentrations of methylene blue . ( C ) Mcg with various concentrations of sulfasalazine . As the concentration of ligand is increased , the ability of VLs to form amyloid fibrils is hindered , as indicated by a decrease in ThT assay fluorescence and inability to detect fibrils with EM . The ligand concentration that effectively inhibited amyloid formation in these assays was 500 μM for methylene blue and 250 μM for sulfasalazine . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 006 Equilibrium dialysis binding of methylene blue and sulfasalazine . Equilibrium dialysis was used to assess the binding constants of methylene blue and sulfasalazine to Mcg . Measured concentrations were fit to the corresponding model equations and their curves were represented as binding and Scatchard plots ( Figure 5 ) ( Scatchard , 1949; Spitzer and McDonald , 1956 ) . The constants were derived from a least squares fit of equations to data and are given in Table 1 . Although both methylene blue and sulfasalazine bind to Mcg , the Scatchard plots indicate that binding proceeds through somewhat different pathways: methylene blue shows positive cooperative binding , signifying at least two sites with different binding constants , while sulfasalazine shows no cooperativity and suggests an additional , non-specific binding site ( Figure 5 ) . The best fit for the sulfasalazine-binding data was achieved using a model for two identical , independent binding sites per VL dimer , followed by non-specific binding . 10 . 7554/eLife . 10935 . 007Figure 5 . Binding of ligands to Mcg VLs . Binding curves ( top ) and Scatchard plots ( bottom ) of ligand binding determined from equilibrium dialysis experiments . Each curve represents binding equations fit to the data by least squares . Binding constants were derived from the fit equations ( see Table 1 ) . Vertical bars represent the standard errors of the mean from independently repeated experiments . [B] , [L] , and [U] are bound , total , and unbound concentrations of ligand in μM . ( A ) Methylene blue binding to Mcg . Rhombs show means for 3 independent experiments performed with 1 . 0 mg/ml Mcg . Circles show means for 5 independent experiments performed with 0 . 5 mg/ml Mcg . Notice the sigmoidal shape of the binding plot and concave shape of the Scatchard plot indicating cooperative binding . ( B ) Sulfasalazine binding to Mcg . Rhombs show means for 3 independent experiments performed with 5 . 0 mg/ml Mcg . Circles show means for 3 independent experiments performed with 2 . 5 mg/ml Mcg . Notice the saturated binding and a down-curving shape of the Scatchard plot indicating one binding constant followed by non-specific binding . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 00710 . 7554/eLife . 10935 . 008Table 1 . Disassociation constants of ligands that bind to the Mcg VL dimer . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 008Ligand<K1> [μM] ( SEM ) <K2> or KNS [μM] ( SEM ) NBinding modelEquations fit ( [Y]: bound ligand , [L]: free ligand , [R]: receptor ) methylene blue207 ( 35 ) 21 ( 4 ) 8Two positively cooperative sites R+L⇔ RL+L ⇔ RL2 ( 1 ) [Y]= ( [L]*[R]/K1 ) * ( 1+ [L]/K2 ) ) ( 2 ) [R]=[R0]/ ( 1+[L]/K1+[L]2/ ( K1*K2 ) ) sulfasalazine698 ( 105 ) 21 ( 8 ) ( non-specific binding ) 6Two equivalent + non-specific sites R+L ⇔ RL ( 3 ) [Y]=[R0]*[L]/ ( K1+[L] ) + KNS*[L]SEM – standard error of the mean; N – number of independent experiments KNS – nonspecific binding Crystal structures of methylene blue and sulfasalazine bound to the VL dimer . In the crystal structures of Mcg with methylene blue and sulfasalazine , the ligands bind at the cavity between the two VL domains ( Figure 6 , Table 2 ) . In the structure of Mcg with methylene blue , one ligand is bound to the A-site of the dimer . This differs from equilibrium dialysis results in solution , which indicate at least two methylene blue binding sites . The structure of Mcg with sulfasalazine indicates two symmetry-related molecules bound in the hydrophobic cavity of the dimer with both ligands simultaneously binding to three sites of the VL dimer cavity . 10 . 7554/eLife . 10935 . 009Figure 6 . Crystal structures of Mcg binding methylene blue ( PDB 5ACM ) and Mcg binding sulfasalazine ( PDB 5ACL ) . ( A ) Mcg and methylene blue . Methylene blue binds the cavity between the VL domains . The sulfate ion binds in the hydrophobic cavity at S36 and may block the secondary methylene blue binding site . ( B ) Mcg and sulfasalazine . Two symmetry-related sulfasalazine molecules bind in the hydrophobic cavity between the VL domains , simultaneously occupying all three sites: A , B , and C . ( C ) Overlay of the structures from A and B for comparison and annotation of A-B-C binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 00910 . 7554/eLife . 10935 . 010Table 2 . Statistics of X-ray data collection and atomic refinement . Values in parentheses are for the outermost shell of data . Each structure was derived from a single crystal . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 010Mcg-methylene blue PDB 5ACMMcg-sulfasalazine PDB 5ACLSpace groupP21P21212Cell dimensions a , b , c ( Å ) 39 . 2 , 31 . 1 , 73 . 631 . 8 , 74 . 6 , 39 . 3α , β , γ ( ° ) 90 , 90 . 1 , 9090 , 90 , 90Resolution ( Å ) 39 . 2-1 . 0 ( 1 . 07 ) 29 . 3-1 . 5 ( 1 . 53 ) Rsym ( % ) 7 . 2 ( 59 . 1 ) 5 . 5 ( 52 . 9 ) I/σ ( I ) 9 . 8 ( 2 . 4 ) 18 . 7 ( 3 . 7 ) Completeness ( % ) 88 . 6 ( 81 . 8 ) 97 . 9 ( 83 . 2 ) Redundancy3 . 5 ( 3 . 5 ) 6 . 9 ( 5 . 9 ) No . reflections259214107913Rwork/Rfree ( % ) 11 . 1/12 . 215 . 9/18 . 7No . atoms Protein1610845Ligand/ion3633Water210134Average B-factors ( Å2 ) 16 . 820 . 4R . m . s deviations Bond lengths ( Å ) 0 . 050 . 02Bond angles ( º ) 3 . 272 . 04Crystallization conditions0 . 2 M NH4Cl , 2 . 2 M ( NH4 ) 2SO4 , 0 . 5 M methylene blue0 . 2 M Tri-potassium citrate , 2 . 2 M ( NH4 ) 2SO4 , 0 . 5 M sulfasalazine Effect of ligand binding on quaternary state . We used analytical ultracentrifugation to assess whether ligand binding shifts the equilibrium of Mcg towards more dimers or whether ligands bind to monomers as well . The wavelengths used for optical density readings of methylene blue ( 550 nm ) and sulfasalazine ( 420 nm ) did not overlap with protein absorbance . Distributions of molecular weights were fit to the absorbance data , thus permitting detection of only the apparent molecular weights of the protein in complex with ligand ( Figure 7 , Table 3 ) . The observed molecular weights indicate that ligands bind only to VL dimers . For methylene blue , the measured molecular weight was slightly larger than expected; therefore , we performed an additional ultracentrifugation meniscus depletion experiment to detect possible monomers . The experiment identified no methylene blue bound to VL monomers . 10 . 7554/eLife . 10935 . 011Figure 7 . Analytical ultracentrifugation data for Mcg with methylene blue and Mcg with sulfasalazine . ( A ) Absorbance and residuals for 80 μM Mcg with 100 μM methylene blue at 22 , 000 rpm . ( B ) Absorbance and residuals for 80 μM Mcg with 150 μM methylene blue at 22 , 000 rpm . ( C ) Dimer meniscus depletion - absorbance and residuals for 80 μM Mcg with 100 μM methylene blue at 35 , 000 rpm . ( D ) Dimer meniscus depletion - absorbance and residuals for 80 μM Mcg with 150 μM methylene blue at 35 , 000 rpm . ( E ) Absorbance and residuals for 200 μM Mcg with 300 μM sulfasalazine at 22 , 000 rpm . ( F ) Absorbance and residuals for 200 μM Mcg with 500 μM sulfasalazine at 22 , 000 rpm . Measurements were taken after samples reached equilibrium and the distribution of protein along the cells did not change . Residuals show the corresponding error of the fit of the model to the data . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 01110 . 7554/eLife . 10935 . 012Table 3 . Apparent VL molecular weights and ligand binding derived from analytical ultracentrifugation . DOI: http://dx . doi . org/10 . 7554/eLife . 10935 . 012Speed ( rpm ) Concentration of Mcg ( μM ) LigandConcentration of ligand ( μM ) Apparent molecular weight ( Da ) Oligomer state22 , 00080methylene blue10027 , 342 ± 268*Dimer + multimer15026 , 507 ± 225*Dimer + multimer35 , 00080methylene blue10023 , 609 ± 212*Dimer15023 , 397 ± 152*Dimer22 , 000200sulfasalazine30021 , 989 ± 571*Dimer50021 , 821 ± 345*Dimer*Standard error .
The identification of ligands that inhibit LC amyloid formation by stabilizing the VL dimer has therapeutic implications for systemic light chain amyloidosis . By utilizing the known ability of ligands to bind the hydrophobic cavity of VL dimers , we prevent dimer disassociation into monomers and thus hinder amyloid formation ( Edmundson et al . , 1984; 1993 ) . A similar approach was used to design therapies for transthyretin-related amyloidosis by stabilizing the tetrameric state of the protein ( Miroy et al . , 1996 ) . Initial screening for ligands that hinder VL amyloid formation revealed four ligands showing this effect , with methylene blue as the strongest binding ligand . A subsequent search in the DrugBank aimed to identify similarly effective ligands that are already approved for medical use . Since the emission spectra of ThT partly overlaps with the absorption spectra of the ligands , both ThT assays and EM analysis were necessary to confirm the ability of the ligands to inhibit amyloid formation . Analysis of electron micrographs and ThT assays indicates that increasing the concentration of methylene blue or sulfasalazine hinders the formation of amyloid fibrils . One of the molecules from our screen , epigallocatechin gallate , was recently suggested to hinder the formation of amyloid fibrils , yet in our experiments it did not show any effect ( Pelaez-Aguilar et al . , 2015 ) . Though both of the ligands bind Mcg and induce amyloid hindering effects , the mechanism of binding slightly differs . Methylene blue binds through positive cooperativity . This result indicates the presence of two binding sites , one strong and one weak , where binding to the second site begins only after the first site is fully occupied . Scatchard plots for sulfasalazine show that the two specific binding sites on Mcg are identical with the same disassociation constant and that this specific ligand binding is followed by non-specific binding . We addressed the difference in the binding mechanisms of methylene blue and sulfasalazine to Mcg in solution by crystal structures and analytical ultracentrifugation . The crystal structure of Mcg with methylene blue shows binding at the A-site of the VL dimer cavity and coordination by the side chains of residues Y34 , Y93 , D97 and F99 . The crystal belongs to space group P21 with one dimer per asymmetric unit , and the symmetry between the two VL domains of Mcg is broken by the presence of a sulfate ion bound to only one of the VL domains ( Figure 6A ) . Analytical ultracentrifugation analysis of the soluble quaternary state indicates the dimer as the most prevalent form of the complex versus monomer without a bound ligand ( Brumshtein et al . , 2014 ) . We also observed a small fraction of oligomers with molecular weights larger than dimers , as confirmed by the meniscus depletion experiment . The meniscus depletion distributes quaternary states along the path length of the sample cell during analytical centrifugation , thus verifying which state accounts for the deviation in apparent molecular weight . The most significant outcome is the complete absence of soluble monomer in complex with the ligand , which verifies our conjecture that the ligands bind and stabilize dimers . In the case of sulfasalazine , to confirm binding specifically to soluble dimers , the analytical ultracentrifugation experiments identified dimers as the only quaternary state of the ligand-bound complex . The reconciliation of our three experiments on the binding of methylene blue to Mcg VLs suggests blocking of the secondary binding site in the crystals . The three experimental findings are: cooperative binding of methylene blue as measured by equilibrium dialysis , binding of the ligand to the A-site in the crystal structure , and the absence of monomeric VL in complex with ligand . Taken together , these observations indicate that the secondary binding site of methylene blue may not be available in the crystalline state , but available in solution . Secondary binding to either the B or C-sites may require conformational changes that are less favorable in crystals or induce the higher-order assembly of complexes larger than dimers . The crystal structure of Mcg with sulfasalazine explains why only one disassociation constant for specific binding was measured . Crystals of the complex belong to space group P21212 with two symmetry-related ligands bound per Mcg dimer , and thus one ligand bound per VL in the asymmetric unit ( Figure 6B ) . Therefore , the equivalence of the two sulfasalazine ligands in the crystal explains why only one disassociation constant was measured by means of equilibrium dialysis . The crystal structure shows that each of the sulfasalazine ligands occupy all three A-B-C-sites simultaneously; each ligand binds to the A and B-sites on one of the VLs composing the dimer , while C-site binding is on the symmetry-related VL . This contrasts with methylene blue , which is bound only to the A-site . Despite differences in the exact orientation of the side chains where the ligands bind , both ligands bind in the cavity of the Mcg dimer ( Figure 6C ) . Although our experiments were performed using only VLs rather than full-length LCs , the ligand-binding site between VLs is maintained in full-length LCs . While full-length LCs are disulfide linked at their CL C-termini , crystal structures indicate that the conformation of VLs is independent of connected constant domains ( Ely et al . , 1989; Terzyan et al . , 2003; Makino et al . , 2007 ) . In full-length LCs , the joining ( J ) segment between VLs and CLs provides the flexibility for VLs to maintain the same hydrophobic cavity as that seen in the VL homo-dimer alone . This indicates the strategy of using ligands to stabilize the VL dimer and hinder amyloid fibril formation may also be effective for full-length homo-dimers . The ability of ligands to hinder the aggregation of VLs into amyloid fibrils by stabilizing VL dimers indicates a promising approach for treatment of systemic light chain amyloidosis disease . While the light chain amino acid sequence differs between patients , the characteristic hydrophobic cavity of the homo-dimer formed by overexpressed LCs is conserved ( Bodi et al . , 2009; Bork et al . , 1994; Edmundson et al . , 1976; Solomon and Weiss , 1995 ) . Although our experiments were performed using a single VL variant , LCs of both λ and κ maintain a similar tertiary structure and hydrophobic cavity ( Qin et al . , 2007; Wetzel , 1994; Brumshtein et al . , 2014; Bellotti et al . , 2000; Fink , 1998; Khurana et al . , 2001 ) . This conserved hydrophobic pocket indicates a possible target for designing medications that inhibit LC amyloidosis . Although methylene blue and sulfasalazine may not bind tightly enough to be directly repurposed as treatments for the disease , these ligands may serve as prototypes for development of effective medications . As indicated by the ability of these molecules to inhibit amyloid fibril formation , this approach may result in alleviated symptoms for systemic light chain amyloidosis patients .
Protein samples were prepared as previously described ( Brumshtein et al . , 2014 ) . In summary , the VL of the pathologic LC homo-dimer Mcg was expressed in E . Coli , purified in denaturing conditions , refolded , and concentrated . Following previous research demonstrating the ability of ligands to bind in the hydrophobic cavity between the two VL domains of the dimer , aromatic and other hydrophobic ligands were screened for an inhibitory effect on the formation of amyloid fibrils ( Edmundson et al . , 1984;1993 ) . Amyloid formation was monitored with Thioflavin T assays and electron microscopy analysis , and we used molecules that inhibited amyloid fibril formation at concentrations below 1 mM to search DrugBank to identify biomedical compounds with a similar structure that would potentially inhibit the formation of amyloid fibrils ( Law et al . , 2014 ) . We selected structures in DrugBank with a similarity threshold of 0 . 3 . Search filters were not applied to any other parameters . We dissolved the identified molecules in either water or 20% DMSO for subsequent use in assays of amyloid formation . Thioflavin ( ThT ) assays of Mcg amyloid formation were performed in acidic conditions as previously described ( Bellotti et al . , 2000 ) . Assays were conducted in the presence of varying concentrations of the ligands ( 1 . 0 , 0 . 50 , 0 . 25 , 0 . 12 , 0 . 05 , and 0 . 03 mM ) at 37°C and constant 300 rpm shaking with Teflon balls with 0 . 125 inch radii as stirrers . ThT fluorescence spectra were recorded at 440/480 nm excitation/emission wavelengths ( Wall et al . , 1999 ) . Samples from the ThT fibril formation assays were diluted with water to 10% v/v and applied onto copper grids with formvar-carbon coating ( Ted Pella , Inc . , Redding , CA , Cat . No . 01810 ) . Negative staining was performed with 2% w/v uranyl acetate and images were collected by means of a Tecnai T12 electron microscope at 120 kV with a Gatan CCD camera . Binding constants of the ligands with Mcg were derived using Rapid Equilibrium Dialysis plates with a membrane cutoff of 8 kDa ( Thermo Scientific Inc . , Cat . No . 90006 ) ( Waters et al . , 2008 ) . Methylene blue at concentrations ranging from ~1–200 μM with 0 . 5 and 1 . 0 mg/ml of Mcg , and sulfasalazine at concentrations ranging from ~100–5000 μM with 2 . 5 and 5 . 0 mg/ml of Mcg were incubated for 72 hours with constant agitation in standard laboratory conditions . Following incubation , samples were collected from the membrane and reference cells and final concentrations were measured by spectrophotometry . Co-crystallization trials were set up with a Mosquito micro-crystallization robot , using a solution of 20 mg/ml Mcg with 1 mM methylene blue or sulfasalazine ligands . All crystals appeared in the presence of sulfate or phosphate anions . Crystals were cryo-protected with 25% glycerol and X-ray data were collected at the 24-ID-C beamline of the Advanced Photon Source ( Mcg-methylene blue ) or with a Rigaku Raxis4++ with a HCT detector ( Mcg-sulfasalazine ) . X-ray diffraction data were processed with XDS package ( Kabsch , 2010 ) . Initial phases were obtained by means of molecular replacement using PDB 3MCG as the initial model and structures were refined with Refmac5 ( Murshudov et al . , 1997 ) . Graphics were rendered with PyMol and refined atomic models were deposited into the Protein Data Bank ( PDB ) ( DeLano , 2002 ) . Sedimentation equilibrium experiments were performed on Mcg in the presence of methylene blue and sulfasalazine . Equilibrium runs were performed at 20°C in a Beckman Optima XL-A analytical ultracentrifuge using absorption optics at 500 nm for methylene blue and 420 nm for sulfasalazine . Samples were at concentrations of 1 mg/ml of Mcg with 100 and 150 μM of methylene blue , and 1 and 2 . 5 mg/ml of Mcg with 300 and 500 μM sulfasalazine . The concentrations of the protein and ligands were chosen to obtain optical density readings in the linear range of the spectrophotometer yet with measurable quaternary state densities of the complexes . Sedimentation equilibrium profiles were measured at speeds of 10 , 000 , 14 , 000 and 22 , 000 rpm in a An-60 Ti rotor . To validate the deviation of the apparent molecular weight of Mcg with methylene blue , a set of experiments analyzing the meniscus depletion of the Mcg dimer were performed at 35 , 000 rpm . The data were fit with a non-linear least squares double exponential fit using the Beckman Origin-based software ( Version 3 . 01 ) ( Cohn and Edsall , 1943; Laue et al . , 1992 ) .
Overproduction of immunoglobulin light chains leads to systemic amyloidosis , a lethal disease characterized by the formation of amyloid fibrils in patients' tissues . Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state . The dimers therefore must disassociate into monomers prior to forming amyloid fibrils . Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer .
Systemic light chain amyloidosis is a disease that occurs when individuals produce too much of an immune protein . The excess protein chains normally exist in the body as individual molecules called “monomers” or in pairs called “dimers , ” and they can readily switch between these two forms . However , the monomers are also prone to forming amyloid fibrils , which are difficult to break down . Amyloid fibrils are often deposited in the heart and kidneys and can lead to organ failure and death . Finding molecules that prevent the formation of amyloid fibrils could help to develop treatments for amyloidosis . Now , Brumshtein , Esswein et al . have screened 27 compounds to identify those that stabilize the dimer form of the protein . This would reduce the number of monomers in the body , and so reduce the number of immune proteins that can form amyloid fibrils . The experiments identified four compounds that could stabilize the dimers , including one called methylene blue . Comparing the chemical structures of these compounds with the structures of drugs approved for medical use identified thirteen drugs . However , follow-up tests showed that only one , called sulfasalazine , reduced the formation of amyloid fibrils . Neither methylene blue nor sulfasalazine is likely to have a strong enough effect to treat amyloidosis , but they may serve as templates for future drug designs .