Collagen IVα345 dysfunction in glomerular basement membrane diseases. II. Crystal structure of the α345 hexamer
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100591
ISSN1083-351X
AutoresSergei P. Boudko, Ryan Bauer, Sergei Chetyrkin, Sergey V. Ivanov, Jarrod A. Smith, Paul Voziyan, Billy G. Hudson,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoOur recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer–trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions. Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer–trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions. Prominent diseases of the glomerular basement membrane (GBM), a specialized form of the extracellular matrix, are diabetic nephropathy (DN), Alport syndrome (AS), and Goodpasture's disease (GP). The morphological abnormalities in the GBM involve structural alterations in collagen IVα345 scaffold, the major GBM component (1Hudson B.G. Reeders S.T. Tryggvason K. Type IV collagen: Structure, gene organization, and role in human diseases. Molecular basis of goodpasture and Alport syndromes and diffuse leiomyomatosis.J. Biol. Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 2Hudson B.G. Tryggvason K. Sundaramoorthy M. Neilson E.G. Alport's syndrome, Goodpasture's syndrome, and type IV collagen.N. Engl. J. Med. 2003; 348: 2543-2556Crossref PubMed Scopus (743) Google Scholar, 3Naylor R.W. Morais M. Lennon R. Complexities of the glomerular basement membrane.Nat. Rev. Nephrol. 2021; 17: 112-127Crossref PubMed Scopus (37) Google Scholar). The mechanisms whereby collagen IV enables normal GBM function or causes GBM abnormalities and dysfunction in disease are unknown. In Pokidysheva et al. (4Pokidysheva E.N. Seeger H. Pedchenko V. Chetyrkin S. Bergmann C. Abrahamson D. Cui Z.W. Delpire E. Fervenza F.C. Fidler A.L. Fogo A.B. Gaspert A. Grohmann M. Gross O. Haddad G. et al.Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.J. Biol. Chem. 2021; 296 (100590)Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), we found that the α345 hexamer, a key connection module within the collagen IVα345 scaffold, is a focal point of bioactivity within the GBM, based on investigation of the Zurich variant that caused AS. To understand how variants, including the Z-variant, in AS cause renal dysfunction, knowledge of the 3D structure of the α345 hexamer is critical. Moreover, this knowledge is also critical to understanding renal dysfunction in GP and DN and development of therapies. Therefore, we solved a crystal structure of the α345NC1 hexamer, a goal that has been pursued by scientists for several decades. The crystal structure revealed features critical for GBM function and in pathogenesis of AS and GP, and, potentially, DN, thus providing a framework for the development of therapies. After decades of attempts to isolate and crystallize the α345 hexamer, we developed a recombinant single-chain NC1 trimer technology (5Pedchenko V. Bauer R. Pokidysheva E.N. Al-Shaer A. Forde N.R. Fidler A.L. Hudson B.G. Boudko S.P. A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.J. Biol. Chem. 2019; 294: 7968-7981Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) and used it to define the arrangement of chains and solve the crystal structure of the recombinant α345 hexamer. The collagen IVα345 scaffold, composed of the α3, α4, and α5 chains, is a major constituent of the GBM. The α345 hexamer can be extracted from the GBM using collagenase treatment (6Boudko S.P. Danylevych N. Hudson B.G. Pedchenko V.K. Basement membrane collagen IV: Isolation of functional domains.Methods Cel. Biol. 2018; 143: 171-185Crossref PubMed Scopus (27) Google Scholar). We previously determined the equimolar composition of α3, α4, and α5 chains in the α345 hexamers isolated from the GBM (7Borza D.B. Bondar O. Todd P. Sundaramoorthy M. Sado Y. Ninomiya Y. Hudson B.G. Quaternary organization of the goodpasture autoantigen, the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains.J. Biol. Chem. 2002; 277: 40075-40083Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) (Fig. 1A). In the same study, we also found the presence of α3-α5 and α4-α4 covalently linked dimers (7Borza D.B. Bondar O. Todd P. Sundaramoorthy M. Sado Y. Ninomiya Y. Hudson B.G. Quaternary organization of the goodpasture autoantigen, the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains.J. Biol. Chem. 2002; 277: 40075-40083Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), where chain monomers from opposite trimers were connected by sulfilimine bonds (8Vanacore R. Ham A.J. Voehler M. Sanders C.R. Conrads T.P. Veenstra T.D. Sharpless K.B. Dawson P.E. Hudson B.G. A sulfilimine bond identified in collagen IV.Science. 2009; 325: 1230-1234Crossref PubMed Scopus (154) Google Scholar) (Fig. 1B). However, the exact chain arrangement within the hexamer is unknown. Taking into account our previous findings, there are only three possible chain arrangements in the α345 hexamers: (1) α345-to-α345, (2) α543-to-α543, and (3) α343-to-α545 (Fig. 1C). We tested these possibilities experimentally using the single-chain NC1 trimer technology (5Pedchenko V. Bauer R. Pokidysheva E.N. Al-Shaer A. Forde N.R. Fidler A.L. Hudson B.G. Boudko S.P. A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.J. Biol. Chem. 2019; 294: 7968-7981Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) developed and verified for the α121 hexamer. This technology allows to define composition and orientation of the chains in the trimer (Fig. 2). The α345, α543, α343, and α545 NC1 single-chain NC1 trimers carrying the signal peptide for secretion (Fig. S1) were transiently expressed in expiCHO cells. Total cell lysates and media were analyzed for the presence of protein of interest using Western blotting (Fig. S2). Only α345 and α545 constructs were detected in the media, whereas α543 and α343 were exclusively trapped within the cells, indicating misfolding problem. Although α545 was partially secreted to the medium, the required partner, α343, was trapped within the cells. Coexpression of α343 and α545 did not rescue secretion of α343. Collectively, the single-chain α345 NC1 trimer represents native composition and orientation of chains. This is also supported by previous studies where association of individual α4 or α5 NC1 monomers with the α3 chain was selectively blocked by the mAbs (7Borza D.B. Bondar O. Todd P. Sundaramoorthy M. Sado Y. Ninomiya Y. Hudson B.G. Quaternary organization of the goodpasture autoantigen, the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains.J. Biol. Chem. 2002; 277: 40075-40083Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The single-chain α345 NC1 trimer produced recombinantly in stably transfected HEK293 cells was purified to homogeneity (Fig. S3) and crystallized in the presence of sodium chloride. The crystal structure was solved at the 1.76 Å resolution with a single polypeptide chain per asymmetric unit (Table S1). It has the designed orientation of α chains in the order of α3-to-α4-to-α5 (Figs. 2 and 3). The atomic structure is homologous to the crystal structure of the α121 NC1 domain (5Pedchenko V. Bauer R. Pokidysheva E.N. Al-Shaer A. Forde N.R. Fidler A.L. Hudson B.G. Boudko S.P. A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.J. Biol. Chem. 2019; 294: 7968-7981Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) (Fig. 3), which is also homologous to all reported crystal structures of tissue extracted from the human and bovine α121 NC1 domain (9Sundaramoorthy M. Meiyappan M. Todd P. Hudson B.G. Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes.J. Biol. Chem. 2002; 277: 31142-31153Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Than M.E. Henrich S. Huber R. Ries A. Mann K. Kuhn K. Timpl R. Bourenkov G.P. Bartunik H.D. Bode W. The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6607-6612Crossref PubMed Scopus (104) Google Scholar, 11Vanacore R.M. Shanmugasundararaj S. Friedman D.B. Bondar O. Hudson B.G. Sundaramoorthy M. The alpha1.alpha2 network of collagen IV. Reinforced stabilization of the noncollagenous domain-1 by noncovalent forces and the absence of Met-Lys cross-links.J. Biol. Chem. 2004; 279: 44723-44730Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Least-square superpositions of whole α345 and α121 trimers and individual chains revealed no significant variations between corresponding Cα atoms (overall r.m.s.d. 0.67 Å) (Table S2). Remarkably, α4 chain has the highest r.m.s.d. value of 2.03 Å when superposed with the α2 chain, although still in the range for highly homologous proteins (12Kufareva I. Abagyan R. Methods of protein structure comparison.Methods Mol. Biol. 2012; 857: 231-257Crossref PubMed Scopus (247) Google Scholar). Most of the structural difference is due to presence of two extra residues, which is unique for α4 chain, within the bottom loop, making a contact with the adjacent α5 chain (Fig. 3). Superposition of α3 and α5 chains with corresponding α1 chains has only 0.55 Å and 0.54 Å r.m.s.d. values, which are typical even for identical proteins (12Kufareva I. Abagyan R. Methods of protein structure comparison.Methods Mol. Biol. 2012; 857: 231-257Crossref PubMed Scopus (247) Google Scholar). Structures of α3 and α5 chains in the α345 NC1 trimer are also identical to the crystal structures observed in α3 and α5 homotrimers/homohexamers, r.m.s.d. of 0.55 to 0.60 Å for α3 and 0.58 Å for α5 (Table S3). Nevertheless, NC1 domains demonstrate sufficient plasticity by forming α2 homotetramer/homo-octamer and α4 homohexamer/homododecamer upon crystallization (13Casino P. Gozalbo-Rovira R. Rodriguez-Diaz J. Banerjee S. Boutaud A. Rubio V. Hudson B.G. Saus J. Cervera J. Marina A. Structures of collagen IV globular domains: Insight into associated pathologies, folding and network assembly.IUCrJ. 2018; 5: 765-779Crossref PubMed Scopus (6) Google Scholar), although for the price of ∼20% and ∼10% of their inner sequences being unstructured. Superposition of the α4 structure of the α345 trimer with the structured part of α4 in the artefactual α4 homohexamer/homododecamer has r.m.s.d. values in the range 4.50 to 4.74 Å, pointing to significant variations even for the structured part (Table S3). High homology and identity of the structures of α3, α4, and α5 chains in the α345 NC1 trimer with the α1 and α2 chains in the NC1 trimer and with α3 and α5 chains in homotrimers/homohexamers also verifies the correct design of the artificial linkers connecting α3-to-α5 and α4-to-α5. All C4 subdomain linkers, native and artificially introduced, are well structured (Fig. 3C), related by a pseudohexagonal symmetry and have comparable atomic displacement factors (Fig. S8 in Supporting Section 3), which further verifies the design of artificial linkers. Analysis of the crystal structure of the single-chain α345 NC1 trimer reveals an unexpected pairing of chains at the hexamer interface. In the crystal structures of the α121 NC1, the pairing follows the rule even–even and odd–odd, that is, α2–α2 and α1–α1, which is consistent with covalent sulfilimine cross-linking of these pairs (8Vanacore R. Ham A.J. Voehler M. Sanders C.R. Conrads T.P. Veenstra T.D. Sharpless K.B. Dawson P.E. Hudson B.G. A sulfilimine bond identified in collagen IV.Science. 2009; 325: 1230-1234Crossref PubMed Scopus (154) Google Scholar). Based on sulfilimine cross-linking analysis of the GBM α345 hexamer, the expected chain pairs were α3–α5 and α4–α4 (7Borza D.B. Bondar O. Todd P. Sundaramoorthy M. Sado Y. Ninomiya Y. Hudson B.G. Quaternary organization of the goodpasture autoantigen, the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains.J. Biol. Chem. 2002; 277: 40075-40083Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), although in the crystal structure, we observed α3–α3 and α4–α5 pairs. Thus, the present crystal structure of the α345 hexamer demonstrates a labile nature of a trimer–trimer orientation in the hexamer before sulfilimine cross-linking. Presumably, during crystallization process, only one particular form was selected as compatible with a given crystal packing. In support of this labile nature of the hexamer are the crystal structures of α3 and α5 homohexamers, which demonstrated alternative pairs of chains in hexamers (13Casino P. Gozalbo-Rovira R. Rodriguez-Diaz J. Banerjee S. Boutaud A. Rubio V. Hudson B.G. Saus J. Cervera J. Marina A. Structures of collagen IV globular domains: Insight into associated pathologies, folding and network assembly.IUCrJ. 2018; 5: 765-779Crossref PubMed Scopus (6) Google Scholar). How nature selects one particular orientation before covalent cross-linking by sulfilimine bonds remains to be explored. In conclusion, our single-chain NC1 trimer method allowed for determination of the α345 hexamer crystal structure, a major goal that has been pursued by scientists for decades. Despite rotational mismatch of the crystallized hexamer, we discovered a set of twelve Cl- ions at the trimer–trimer interface (Fig. 4) having the same geometry as in the α121 hexamer (5Pedchenko V. Bauer R. Pokidysheva E.N. Al-Shaer A. Forde N.R. Fidler A.L. Hudson B.G. Boudko S.P. A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.J. Biol. Chem. 2019; 294: 7968-7981Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). All 12 Cl- at the trimer–trimer interface have comparable electron densities (Fig. S4) and atomic displacement factors in the range from 19.1 to 19.7 Å2, like the core residues of the polypeptide chain (Fig. 8). Together, the 12 ions form a chloride ring at the hexamer interface (Fig. 5). Analogously to the Cl- ions in the α121 hexamer, these ions form two structurally different groups (Figs. 5, S4, and Table S4). Geometry and residue specificity of Cl- coordination are identical between α345 and α121, with only one exception, that is, two-thirds of group 1 ions (four ions per hexamer) are not coordinated by the salt bridge to the arginine residue of the opposite trimer, rather identical arginine within the same trimer coordinates respective ions (Cl- ions #1–2). Potentially, the absence of these salt bridges might impact the hexamer stability of the crystallized form. The other two Cl- ions (#3) in group 1 have 'classical' geometry observed in the α121 hexamer with the arginine residues from the opposite trimer involved in the coordination (5Pedchenko V. Bauer R. Pokidysheva E.N. Al-Shaer A. Forde N.R. Fidler A.L. Hudson B.G. Boudko S.P. A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.J. Biol. Chem. 2019; 294: 7968-7981Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar).Figure 5The α345 hexamer crystal structure reveals a ring of chloride ions that coordinate the trimer–trimer interface. A, the crystal structure of the α345 hexamer is shown as a backbone wireframe (left). Individual chains are indicated by different colors as shown on the right. EA and EB loops in the α3 chain are shown in yellow and orange, respectively. 12 chloride ions (blue spheres) form a Cl- ring at the interface between two α345 trimers (left and right). B, chloride ion coordination for group 1 chloride. Group 1 chloride are responsible for introducing intramolecular salt bridges utilizing chloride coordinating Arg (R76, R300, and R525 in α3, α4, and α5 respectively). Coloring for group 1 chloride is blue shown as blue spheres, while carbon atom coloring in NC1 chains is light red for α3, light blue for α4, and light green for α5. C, chloride ion coordination for group 2 chloride. Group 2 chloride are responsible for directly bridging trimeric protomers in the collagen IV hexamer, and unlike group 1 chloride, are available for interaction with PEG molecules (PG4 and PGE). Coloring for group 2 chloride is cyan, while carbon atom coloring in PEG molecules (PG4 and PGE) is white. Chloride interactions from one NC1 molecule are shown as white dashes, while interactions from the opposite molecule are shown as black dashes. Carbon atom coloring for NC1 chains is the same as above.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chloride ions of group 2 are located in pockets and also interact with PEG molecules used for crystallization (Fig. 6C and Table S4). Although those interactions are nonspecific and weak, they point to possibility to modulate chloride binding and the hexamer assembly by specifically developed agents, which might become drugs. In summary, like the α121 hexamer, the α345 hexamer possesses two groups of chloride ions at the trimer–trimer interface forming a 12-ion ring critical for hexamer assembly and stability. The crystal structure of the α345 hexamer reveals several crevices, pockets, and inner cavities, which are large enough to accommodate small molecules (Fig. 6, A and B). Under crystallization conditions used, we observe not only chloride ions, which are physiologically relevant and critical for the hexamer assembly, but also multiple PEG molecules. As discussed earlier, the chloride ions of group 2 are sitting at the bottom of pockets, which are also occupied by PEG molecules. Chloride ions of group 2 are localized in small inner cavities, which additionally contain several structured water molecules (Fig. 6C). The central inner cavity going from one trimer to another through the hexamer interface accommodates multiple structured PEG molecules but would accommodate much larger molecules if present during protein folding or the hexamer assembly. We also found crevices between chains and between C4 subdomains within each chain. The crevices between chains are wider and occupied by PEG molecules (Fig. 6B). The crevices are close enough to the inner cavity and potentially there is a communication between these structures under physiological conditions. In support of this molecular channel is the presence of PEG molecules in the inner cavity, although the protein used for crystallization has been already in the hexamer form (addressed below). Thus, even for the fully assembled hexamer there is a mechanism of penetration of ligands into the inner cavity. The outer surface of the α345 hexamer represents a complex landscape with multiple hills and valleys. We found multiple PEG molecules interacting with the surface and some of them having contacts with two adjacent chains (Fig. S5). There are surface-exposed loops on the α345 hexamer (Fig. 7) that encompass the EA and EB regions of GP immunoreactivity (14Netzer K.O. Leinonen A. Boutaud A. Borza D.B. Todd P. Gunwar S. Langeveld J.P. Hudson B.G. The Goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain.J. Biol. Chem. 1999; 274: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies.J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The loops are designated herein as hypoepitopes as they are not recognized by GP autoantibodies but can undergo a conformational transition into neoepitopes that bind the antibodies (16Pedchenko V. Bondar O. Fogo A.B. Vanacore R. Voziyan P. Kitching A.R. Wieslander J. Kashtan C. Borza D.B. Neilson E.G. Wilson C.B. Hudson B.G. Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis.N. Engl. J. Med. 2010; 363: 343-354Crossref PubMed Scopus (244) Google Scholar, 17Calvete J.J. Revert F. Blanco M. Cervera J. Tarrega C. Sanz L. Revert-Ros F. Granero F. Perez-Paya E. Hudson B.G. Saus J. Conformational diversity of the Goodpasture antigen, the noncollagenous-1 domain of the alpha3 chain of collagen IV.Proteomics. 2006; 6: S237-S244Crossref PubMed Scopus (9) Google Scholar). The EA and EB loops (Fig. 7) demonstrate elevated mean square displacement values (Fig. 8), which reflect increased dynamic mobility of the loops. The EA loop of α5 chain is involved in crystal packing, thus having relatively lower B values (Fig. 8). Mutation analysis showed evolutionary pressure on the loop sequences, particularly on the EA loop in α2-α5 chains (Fig. S6), supporting the functional importance of these loops. One of the crevices is located between EA and EB hypoepitope loops forming loop-crevice-loop (LCL) regions at the apexes of the α345 hexamer and is juxtaposed with the T-cell receptor epitopes (18Ooi J.D. Petersen J. Tan Y.H. Huynh M. Willett Z.J. Ramarathinam S.H. Eggenhuizen P.J. Loh K.L. Watson K.A. Gan P.Y. Alikhan M.A. Dudek N.L. Handel A. Hudson B.G. Fugger L. et al.Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells.Nature. 2017; 545: 243-247Crossref PubMed Scopus (134) Google Scholar, 19Phelps R.G. Rees A.J. The HLA complex in Goodpasture's disease: A model for analyzing susceptibility to autoimmunity.Kidney Int. 1999; 56: 1638-1653Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 20Xie L.J. Cui Z. Chen F.J. Pei Z.Y. Hu S.Y. Gu Q.H. Jia X.Y. Zhu L. Zhou X.J. Zhang H. Liao Y.H. Lai L.H. Hudson B.G. Zhao M.H. The susceptible HLA class II alleles and their presenting epitope(s) in Goodpasture's disease.Immunology. 2017; 151: 395-404Crossref PubMed Scopus (12) Google Scholar).Figure 8Mean square displacement (B values) of Cα atoms. B values for native and artificial linkers are comparable as depicted by green and red diamonds, respectively. B values for EA and EB hypoepitopes are shown as yellow and orange circles. Residue positions are marked with colored bars: light red for α3, light blue for α4, and light green for α5 chains. Borders between C41 and C42 subdomains are depicted as vertical lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Z-appendage is an 8-residue C-terminal extension of the native primary structure of the α3 chain of collagen IV. It is located at the apex of the α3NC1 monomer, in juxtaposition with EA and EB hypoepitope loops (Fig. 9). To assess Z-appendage flexibility, a molecular dynamics (MD) simulation was performed on the appendage in the context of a model of the α3 chain NC1 monomer. To sample all possible orientations of the appendage, 1000 initial conformations were originally generated by high-temperature MD. Each of those was extended for an additional 1 ns of simulation time at the physiological temperature, resulting in 1 μs of total MD sampling. Cysteines were reduced. Clustering analysis of the Z-appendage residues from all 1000 trajectories (using a 7 Å r.m.s.d. cutoff) revealed 134 conformational families. Within two of those 134 clusters, we found multiple conformations of the Z-appendage that had the cysteine residue positioned adjacent to the EA or EB loop disulfides. Two such conformations are shown in Figure 9, B and C. In these two conformations, the mutant cysteine is near the WT EA/EB cysteines that form an intraloop disulfide. These conformations suggest that the appendage cysteine residue may form alternative disulfides. Interference with disulfide formation and disturbing other interactions within the monomer can lead to a conformational change of the monomer and influence assembly of the hexamer. In conclusion, the Z-appendage can assume multiple conformations and its free thiol group can participate in a number of reactions including those with EA and EB epitope loops. Solving the crystal structure of the α345 hexamer allowed for 3D mapping of known Alport variants. The maps of Alport variants in the α3, α4, and α5 chains of collagen IV and their localizations within 3D structures of the α3, α4, and α5NC1 domains are shown in Figure 10 (for additional details, see Figs. S7–S9). The analysis reveals two classes of NC1 variants, that is, truncating and nontruncating. Potentially, both classes are amenable to protein replacement therapy and the nontruncating class also presents a possibility for development of small-molecule therapies. The descriptions for each variant are provided in the top part of Figs. S7–S9, according to the human genome variation society nomenclature (21den Dunnen J.T. Dalgleish R. Maglott D.R. Hart R.K. Greenblatt M.S. McGowan-Jordan J. Roux A.F. Smith T. Antonarakis S.E. Taschner P.E. HGVS Recommendations for the description of sequence variants: 2016 Update.Hum. Mutat. 2016; 37: 564-569Crossref PubMed Scopus (861) Google Scholar). The Zurich variant of the α3NC1 domain stands out among other Alport variants as it results in a C-terminal extension of protein polypeptide chain producing an 8-amino acid Z-appendage as shown in Fig. S7; a variant analogous to the Zurich variant producing a 74-amino acid appendage has been identified in α5NC1 (Fig. S9). The α345 hexamer possesses multiple surface-exposed lysine (Lys) and arginine (Arg) residues (Fig. 11) that can be targeted by hyperglycemia-derived reactive carbonyl products to form stable adducts that underlie DN pathogenesis, including Lys–Lys and Lys–Arg crosslinks (22Thorpe S.R. Baynes J.W. Maillard reaction products in tissue proteins: New products and new perspectives.Amino Acids. 2003; 25: 275-281Crossref PubMed Scopus (441) Google Scholar). There are 78 surface-exposed Lys and Arg side chains in the hexamer. Importantly, six of these residues in the α3NC1 domain and four in the α5NC1 domain are adjacent to or located on the respective EA and EB hypoepitopes (Fig. 11). The crystal structure of the α345 hexamer provided a framework to interpret a role that the Z-appendage, a representative AS variant, played in AS and as a possible structural risk factor for GP, as described in Pokidysheva et al. (4Pokidysheva E.N. Seeger H. Pedchenko V. Chetyrkin S. Bergmann C. Abrahamson D. Cui Z.W. Delpire E. Fervenza F.C. Fidler A.L. Fogo A.B. Gaspert A. Grohmann M. Gross O. Haddad G. et al.Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.J. Biol. Chem. 2021; 296 (100590)Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The crystal structure of the hexamer revealed a ring of 12 chloride ions that, together with up to six sulfilimine bonds, stabilizes the hexamer structure (Fig. 12A). Th
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