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• W2110231726 abstract "Metaphyseal chondrodysplasia type Schmid (MCDS) is caused by mutations in COL10A1that are clustered in the carboxyl-terminal non-collagenous (NC1) encoding domain. This domain is responsible for initiating trimerization of type X collagen during biosynthesis. We have built a molecular model of the NC1 domain trimer based on the crystal structure coordinates of the highly homologous trimeric domain of ACRP30 (adipocyte complement-related protein of 30 kDa or AdipoQ). Mapping of the MCDS mutations onto the structure reveals two specific clusters of residues as follows: one on the surface of the monomer which forms a tunnel through the center of the assembled trimer and the other on a patch exposed to solvent on the exterior surface of each monomeric unit within the assembled trimer. Biochemical studies on recombinant trimeric NC1 domain show that the trimer has an unusually high stability not exhibited by the closely related ACRP30. The high thermal stability of the trimeric NC1 domain, in comparison with ACRP30, appears to be the result of a number of factors including the 17% greater total buried solvent-accessible surface and the increased numbers of hydrophobic contacts formed upon trimerization. The 27 amino acid sequence present at the amino terminus of the NC1 domain, which has no counterpart in ACRP30, also contributes to the stability of the trimer. We have also shown that NC1 domains containing the MCDS mutations Y598D and S600P retain the ability to homotrimerize and heterotrimerize with wild type NC1 domain, although the trimeric complexes formed are less stable than those of the wild type molecule. These studies suggest strongly that the predominant mechanism causing MCDS involves a dominant interference of mutant chains on wild type chain assembly. Metaphyseal chondrodysplasia type Schmid (MCDS) is caused by mutations in COL10A1that are clustered in the carboxyl-terminal non-collagenous (NC1) encoding domain. This domain is responsible for initiating trimerization of type X collagen during biosynthesis. We have built a molecular model of the NC1 domain trimer based on the crystal structure coordinates of the highly homologous trimeric domain of ACRP30 (adipocyte complement-related protein of 30 kDa or AdipoQ). Mapping of the MCDS mutations onto the structure reveals two specific clusters of residues as follows: one on the surface of the monomer which forms a tunnel through the center of the assembled trimer and the other on a patch exposed to solvent on the exterior surface of each monomeric unit within the assembled trimer. Biochemical studies on recombinant trimeric NC1 domain show that the trimer has an unusually high stability not exhibited by the closely related ACRP30. The high thermal stability of the trimeric NC1 domain, in comparison with ACRP30, appears to be the result of a number of factors including the 17% greater total buried solvent-accessible surface and the increased numbers of hydrophobic contacts formed upon trimerization. The 27 amino acid sequence present at the amino terminus of the NC1 domain, which has no counterpart in ACRP30, also contributes to the stability of the trimer. We have also shown that NC1 domains containing the MCDS mutations Y598D and S600P retain the ability to homotrimerize and heterotrimerize with wild type NC1 domain, although the trimeric complexes formed are less stable than those of the wild type molecule. These studies suggest strongly that the predominant mechanism causing MCDS involves a dominant interference of mutant chains on wild type chain assembly. Type X collagen is a short chain, homotrimeric collagen (α1(X)3) expressed specifically by hypertrophic chondrocytes in the endochondral growth plate (1Schmid T.M. Linsenmayer T.F. J. Cell Biol. 1985; 100: 598-605Crossref PubMed Scopus (350) Google Scholar). The expression of type X collagen is also re-activated during fracture repair and in osteoarthritis (2Grant W.T. Wang G.J. Balian G. J. Biol. Chem. 1987; 262: 9844-9849Abstract Full Text PDF PubMed Google Scholar, 3Girkontaite I. Frischholz S. Lammi P. Wagner K. Swoboda B. Aigner T. Von der Mark K. Matrix Biol. 1996; 15: 231-238Crossref PubMed Scopus (189) Google Scholar, 4Thomas J.T. Cresswell C.J. Rash B. Hoyland J. Freemont A.J. Grant M.E. Boot-Handford R.P. Biochem. Soc. Trans. 1991; 19: 804-808Crossref PubMed Scopus (9) Google Scholar). The human α1(X) collagen chain consists of a short amino-terminal non-collagenous domain 2 of 37 amino acids followed by a triple helix-forming collagenous domain with 154 Gly-X-Y repeats and a carboxyl-terminal non-collagenous domain (NC1) 1The abbreviations used are: NC1, non-collagenous carboxyl-terminal domain of type X collagen; MCDS, metaphyseal chondrodysplasia type Schmid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; nt, nucleotide(s).1The abbreviations used are: NC1, non-collagenous carboxyl-terminal domain of type X collagen; MCDS, metaphyseal chondrodysplasia type Schmid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; nt, nucleotide(s). of 161 amino acids (5Thomas J.T. Cresswell C.J. Rash B. Nicolai H. Jones T. Solomon E. Grant M.E. Boot-Handford R.P. Biochem. J. 1991; 280: 617-623Crossref PubMed Scopus (79) Google Scholar). The α1(X)3 molecule is thought to assemble into a hexagonal lattice within the extracellular matrix in a fashion similar to that of type VIII collagen (6Kwan A.P.L. Cummings C.E. Chapman J.A. Grant M.E. J. Cell Biol. 1991; 114: 597-604Crossref PubMed Scopus (150) Google Scholar, 7Frischholz S. Beier F. Girkontaite I. Wagner K. Poschl E. Turnay J. Mayer U. von der Mark K. J. Biol. Chem. 1998; 273: 4547-4555Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Type X collagen is part of a family of collagen-like proteins sharing a condensed gene structure, a collagen triple helical domain, and in particular, a highly conserved carboxyl-terminal non-collagenous (NC1-like) domain (8Brass A. Kadler K.E. Thomas J.T. Grant M.E. Boot-Handford R.P. FEBS Lett. 1992; 303: 126-128Crossref PubMed Scopus (67) Google Scholar, 9Petry F. Reid K.B.M. Loos M.J. Eur. J. Biochem. 1992; 209: 129-134Crossref PubMed Scopus (21) Google Scholar). This family includes collagen types X and VIII, C1q component of complement, hibernation proteins (10Takamatsu N. Ohba K. Kondo J. Kondo N. Shiba T. Mol. Cell. Biol. 1993; 13: 1516-1521Crossref PubMed Scopus (86) Google Scholar), cerebellin (11Wada C. Ohtani H. Mol. Brain Res. 1991; 9: 71-77Crossref PubMed Scopus (30) Google Scholar, 12Urade Y. Oberdick J. Molinar-Rode R. Morgan J.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1069-1073Crossref PubMed Scopus (97) Google Scholar), and ACRP30, an abundant serum protein implicated in energy homeostasis and obesity (13Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2722) Google Scholar). The precise function of type X collagen remains to be determined (14Jacenko O. LuValle P.A. Olsen B.R. Nature. 1993; 365: 56-61Crossref PubMed Scopus (180) Google Scholar, 15Rosati R. Horan G.S.B. Pinero G.J. Garofalo S. Keene D.R. Horton W.A. De Crombrugghe B. Behringer R.R. Nat. Genet. 1994; 8: 129-135Crossref PubMed Scopus (136) Google Scholar, 16Kwan K.M. Pang M.K.M. Zhou S. Cowan S.K. Kong R.Y.C. Pfordte T. Olsen B.R. Sillence D.O. Tam P.P.L. Cheah K.S.E. J. Cell Biol. 1997; 136: 459-471Crossref PubMed Scopus (154) Google Scholar), but mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS), an autosomal dominant form of human skeletal dysplasia (17Warman M.L. Abbott M. Apte S.S. Hefferon T. McIntosh I. Cohn D.H. Hecht J.T. Olsen B.R. Francomano C.A. Nat. Genet. 1993; 5: 79-82Crossref PubMed Scopus (214) Google Scholar, 18Wallis G.A. Rash B. Sweetman W.A. Thomas J.T. Super M. Evans G. Grant M.E. Boot-Handford R.P. Am. J. Hum. Genet. 1994; 54: 169-178PubMed Google Scholar). An intriguing finding is that virtually all of the mutations causing MCDS occur within the carboxyl-terminal non-collagenous NC1 domain of type X collagen (Ref.19Wallis G.A. Rash B. Sykes B. Bonaventure J. Maroteaux P. Zabel B. Wynnedavies R. Grant M.E. Boot-Handford R.P. J. Med. Genet. 1996; 33: 450-457Crossref PubMed Google Scholar and references therein). The only two MCDS mutations not found in the NC1 domain affect the putative signal peptide cleavage site upstream of the non-collagenous domain 2 in the molecule (20Ikegewara S. Nakamura K. Nagano A. Haga N. Nakamura Y. Hum. Mutat. 1997; 9: 131-135Crossref PubMed Scopus (36) Google Scholar). The role of the carboxyl-terminal non-collagenous domain of most collagens (including type X) in initiating intracellular α-chain selection, assembly, and helix formation is well established (Refs. 8Brass A. Kadler K.E. Thomas J.T. Grant M.E. Boot-Handford R.P. FEBS Lett. 1992; 303: 126-128Crossref PubMed Scopus (67) Google Scholar and 21Bulleid N.J. Dalley J.A. Lees J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (87) Google Scholar and references therein). Assembly studies based on cell-free translation of recombinant RNA encoding wild type and MCDS transcripts of type X collagen suggest that mutant chains do not interfere with the trimerization of the wild type protein based on SDS-PAGE assays (22Chan D. Weng Y.M. Hocking A.M. Golub S. Mc Quillan D.J. Bateman J.F. J. Biol. Chem. 1996; 271: 13566-13572Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These findings have led to the suggestion that the phenotype of MCDS is best explained by haplo-insufficiency. In support of this hypothesis, in one individual with MCDS, only wild type mRNA for type X collagen could be detected in the growth plate, suggesting that the mutant transcript is unstable and rapidly degraded (23Chan D. Weng Y.M. Graham H.K. Sillence D.O. Bateman J.F. J. Clin. Invest. 1998; 101: 1490-1499Crossref PubMed Google Scholar). However, haplo-insufficiency is in discord with the clustering of both point and frameshift mutations in the NC1 domain of type X collagen; in haplo-insufficiency one would expect to find MCDS-causing frameshift mutations to be randomly distributed through the gene (19Wallis G.A. Rash B. Sykes B. Bonaventure J. Maroteaux P. Zabel B. Wynnedavies R. Grant M.E. Boot-Handford R.P. J. Med. Genet. 1996; 33: 450-457Crossref PubMed Google Scholar). The non-random clustering of MCDS mutations in the NC1 domain is more consistent with a mechanism involving dominant interference in which the mutant chains retain the ability to trimerize. It is possible that previous investigations have failed to detect trimerization of chains containing MCDS mutations due to the harsh assay conditions, such as SDS-PAGE, that have been employed. The crystal structure of the NC1-like domain of ACRP30, a protein closely related to type X collagen, has been recently solved at 2.1-Å resolution (24Shapiro L. Scherer P.E. Curr. Biol. 1998; 8: 335-338Abstract Full Text Full Text PDF PubMed Google Scholar). We have therefore performed molecular modeling of the NC1 domain, based on the ACRP30 crystal structure coordinates, to gain greater insight into the structural basis of MCDS. The model reveals that the MCDS mutations are localized to two specific regions of the folded monomeric NC1 domain and that many of the MCDS mutations may not totally abolish the ability of affected chains to trimerize. Mutagenesis experiments, based on information obtained from the model, demonstrate that NC1 domains containing specific MCDS mutations retain the ability to form trimers and provide an explanation for the unusually high thermal stability exhibited by the assembled NC1 trimer. The amino acid sequence of the NC1 domain of human type X collagen was used to probe EMBL, GenBankTM, SwissProt and PIR data bases as well as data base updates using the advanced BLAST 2.0 search (25Pearson W.R. Methods Enzymol. 1990; 183: 63-98Crossref PubMed Scopus (1643) Google Scholar). We used a cut-off expectancy value (E) of 1 (such that no more than 1 match is expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (26Karlin S. Altschul S.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2264-2268Crossref PubMed Scopus (1171) Google Scholar)) and a gapped alignment using the Blosum 62 matrix (27Henikoff S. Henikoff J.G Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10915-10919Crossref PubMed Scopus (4284) Google Scholar). Multalin (28Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4275) Google Scholar) was used to re-align the resulting 25 sequences using the Blosum 62 matrix using a penalty of 12 for gap opening and 2 for gap extending. None of the resulting aligned sequences have three-dimensional structures in the Brookhaven data base. All molecular modeling was performed on an R5000 O2 Silicon graphics workstation using QUANTA® and CHARMm 23.1® programs. The three–dimensional model of the NC1 domain of type X collagen was built based on the coordinates of human ACRP30. 2L. Shapiro, personal communication from Albert Einstein College of Medicine, New York. The file containing the coordinates was rewritten manually to make a Protein Data Base file format that could be imported by QUANTA®. A homology model was built by copying the coordinates of the backbone of the ACRP30 trimer and the coordinates of identical residues in the type X collagen NC1 domain. The remaining side chains were built in the Protein Design module using the Ponder and Richards' rotamer library. Disordered loops in the crystal structure of ACRP30 (loop G-H in M1 and loops G-H and A-A′ in M2) were modeled where possible by overlaying the intact loop of a corresponding part of one of the other two monomers. This left a gap between Glu658 to Gly661 (corresponding to Asp224 to Gly 228 in ACRP30) in each monomer. The resulting trimer of the NC1 domain of type X collagen was energy-minimized (using the steepest descents followed by Newton-Raphson algorithm) to gradient convergence (<0.01 root mean square) removing bad steric and electrostatic contacts. The water molecules from the crystal structure were overlaid, and those internal to the trimer were incorporated into the model. The Protein Health module in QUANTA was used to check the integrity of the model using a Ramachandran map and to identify buried hydrophilic or exposed hydrophobic residues. The solvent-accessible surfaces of monomers and assembled trimers were calculated to estimate the surface area buried upon trimerization. The local environment of each residue on the interfaces was examined to locate those residues important for the trimer structure. The interfaces are not symmetric, because of a slight twist and stagger to the arrangement of the β-sandwich monomers (24Shapiro L. Scherer P.E. Curr. Biol. 1998; 8: 335-338Abstract Full Text Full Text PDF PubMed Google Scholar). Particular attention was given to the position of those residues in the NC1 model trimer that differed in ACRP30 and related proteins (see Fig.1).Figure 1Sequence alignment of C1q–like domains with a BLAST 2.0 Evalue of less than 0.015. BLAST 2.0 was used to probe the data bases with the NC1 domain of type X collagen sequence and retrieved 58 sequences with an E value less than 10. After discarding redundancy we aligned 25 sequences in Multalin using the Blosum 62 matrix and default penalty values. Those residues in red are conserved throughout the alignment; those in blue are conserved in at least 70% of sequences, and those highlighted in yellow show the MCDS point mutations. β-Strand topology is taken from ACRP30 and TNF nomenclature; ca1a_human, human type X collagen (Swiss-Prot accession number Q03692); ca1a_bovin, bovine type X collagen (Swiss-Prot accession number P3206); ca1a_mouse, murine type X collagen (Swiss-Prot accession number Q00780); ca1a_chick, gallus gallus type X collagen (Swiss-Prot accession number P08125); ca28_mouse, murine α2 type VIII collagen (Swiss-Prot accession number P25318); ca28_human, human α2 type VIII collagen (Swiss-Prot accession number P25067); ca18_rabbit, rabbit α1 type VIII collagen (Swiss-Prot accession number P14282); ca18_human, human α1 type VIII collagen (Swiss-Prot accession number P27658); ca18_mouse, murine α1 type VIII collagen (Swiss-Prot accession number Q00780); acr3_human, human adipocyte complement-related protein (Swiss-Prot accession number Q15848); acr3_mouse, murine adipocyte complement-related protein (Swiss-Prot accession number Q60994); cole_lepma, lepomis macrochirusinner ear-specific collagen (Swiss-Prot accession number P98085); hp27_tamas, chipmunk hibernation-associated protein (Swiss-Prot accession number Q06577); hp25_tamas, chipmunk hibernation-associated protein (Swiss-Prot accession number Q06576); hp20_tamas, chipmunk hibernation-associated protein (Swiss-Prot accession number Q06575); c1qc_mouse, murine C1q C chain (Swiss-Prot accession number Q02105); c1qc_human, human C1q C chain (Swiss-Prot accession number P02747); c1qb_mouse, murine C1q B chain (Swiss-Prot accession number P14106); c1qb_rat, rat C1q B chain (Swiss-Prot accession number P31721); c1qb_human, human C1q B chain (Swiss-Prot accession number P02746); c1qa_mouse, murine C1q A chain (Swiss-Prot accession number P98086); c1qa_human, human C1q A chain (Swiss-Prot accession number P02745); cerl_rat, rat cerebellin (Swiss-Prot accession number P23436); cerb_human, human cerebellin 1 (Swiss-Prot accession number P23435); multimerin_h, human multimerin (Swiss-Prot accession numberA57384).View Large Image Figure ViewerDownload Hi-res image Download (PPT) DNA constructs were generated by PCR from the lambda genomic COL10A1 clone, HX3 (5Thomas J.T. Cresswell C.J. Rash B. Nicolai H. Jones T. Solomon E. Grant M.E. Boot-Handford R.P. Biochem. J. 1991; 280: 617-623Crossref PubMed Scopus (79) Google Scholar). DNA encoding the entire human type X collagen NC1 domain was amplified using the primers 5′-CCAGGTCAAGCACATATGCCTGAGGGT-3′ (primer A, sense nucleotides 1550–1563), which incorporates an NdeI site (C (A/T)ATG) in-frame with the Met521 codon of the NC1 domain, and 5′-GGGGTGTACTCACATTGGAGCCAC-3′ (primer B, antisense nucleotides 2028–2052), which incorporates the stop codon and 9 base pairs of the 3′-untranslated region. To generate the extended reporter construct, DNA encoding the entire NC1 domain and upstream DNA encoding the 11 most carboxyl-terminal Gly-X-Y repeats of the collagenous domain was generated by PCR. A sense oligonucleotide of sequence 5′-CCAGCTCATATGGCAACTAAGGGCCTC-3′ (nucleotides 1429–1455), which incorporates an NdeI site coding for an in-frame Met codon at the amino terminus of the extended NC1 coding region, was used together with the antisense primer B. To produce the truncated NC1 translation construct, DNA encoding a portion of the NC1 domain from the Met549 codon to the stop codon was generated by PCR using the sense primer 5′-GGGGTAACACATATGCCTGTGTCT-3′ (nucleotides 1632–1656) together with the antisense primer B. To create constructs containing MCDS and related point mutations, site-directed mutagenesis was carried out using the PCR-based single overlap extension procedure described elsewhere (29Ling M.M. Robinson B.H. Anal. Biochem. 1997; 254: 157-178Crossref PubMed Scopus (183) Google Scholar) using oligonucleotides A and B (described above) and mutagenic oligonucleotides (nucleotides 1875–1899) sense, 5′-CCAGGAATATACTTTTTTTCATAC-3′ and antisense, 5′-GTATGAAAAAAAGTATATTCCTGG-3′ for Y598F; sense, 5′-CCAGGAATATACGCTTTTTCATAC-3′ and antisense, 5′-GTATGAAAAAGCGTATATTCCTGG-3′ for Y598A; 5′-CCAGGAATATACGATTTTTCATAC- 3′ and antisense, 5′-GTATGAAAAATCGTATATTCCTGG-3′ for Y598D; and (nucleotides 1881–2004) sense, 5′-ATATACGCTTTTCCATACCACGTG-3′ and antisense, 5′-CACGTGGTATGGAAAAGCGTATAT-3′ for S600P. The resulting PCR fragments were cloned into the T/A cloning vector pCR 2.1 (Invitrogen). Constructs generating sense RNA upon transcription from the T7 promoter were identified by restriction mapping and sequenced. In vitro expression was carried out using the TNT T7 polymerase-coupled rabbit reticulocyte lysate system (Promega). 1 μg of each purified construct was expressed in 40 μl of TNT premix containing 2 μl of [35S]methionine (1000 Ci/mmol, NEN Life Science Products) made up to a final volume of 50 μl with sterile distilled water. Reactions were incubated at 30 °C for 90 min. 5-μl aliquots of in vitro expression reaction were added to 10 μl of sample buffer (50 mm Tris/HCl, pH 6.8 (room temperature), 50% (v/v) glycerol, 0.025% (w/v) bromphenol blue; the concentration of SDS in the sample buffer was either 10% (w/v) or 0.75% (w/v) as indicated under “Results”). Where heating is indicated, samples were overlaid with 20 μl of mineral oil and incubated at the appropriate temperature for 5 min using Perkin-Elmer 480 thermal cycler prior to analysis by SDS-PAGE. All samples were resolved on standard 12 or 16% PAGE gels containing 0.1% SDS according to Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar). Gels were fixed (10% (v/v) acetic acid containing 10% (v/v) methanol) for 30 min and washed twice in 30% (v/v) methanol containing 3% (v/v) glycerol. Gels were dried and exposed to Kodak Biomax Film, or, if quantitation was required, phosphorimaged (Fuji-Bas). In vitro translations containing labeled NC1 domains were subjected to centrifugation at 109,000 ×g for 1 h to remove particulates. 50 μl of the supernatant was then dialyzed against NET buffer overnight (100 mm Tris/HCl, pH 7.4, 100 mm NaCl, 1% (v/v) Tween 20, 1 mm EDTA). 5 μl of hybridoma medium containing the X34 monoclonal antibody, which recognizes a conformation-dependent epitope in the NC1 domain of native type X collagen (31Girkontaite I. Frischholz S. Lammi P. Wagner K. Swoboda B. Aigner T. Von der Mark K. Matrix Biol. 1996; 15: 231-238Crossref PubMed Scopus (183) Google Scholar), was added to the dialyzed translation followed by incubation for 16 h at 4 °C with gentle inversion. Following the incubation, 10 μl of protein A-Sepharose (Amersham Pharmacia Biotech) was added, and the incubation was allowed to proceed for a further 4 h, after which the protein A-Sepharose was recovered by centrifugation at 12,000 × g (control incubations were carried out in the absence of X34). The pelleted protein A-Sepharose was washed in 500 μl of 1× NET buffer three times followed by resuspension in sample buffer containing 1% (v/v) β-mercaptoethanol. Prior to SDS-PAGE analysis, the samples were heated for 2 min at 68 °C. The complete NC1 domain of human type X collagen was aligned with the carboxyl-terminal non-collagenous domain of mouse ACRP30 (Fig. 2). The type X collagen NC1 domain contains an extra 27 residues at the amino terminus that are not present in ACRP30 and a deletion of ACRP30-Tyr219. The globular domain of ACRP30 exhibits 40% identity and 65% similarity with the equivalent region of the type X collagen NC1 domain. Overlaying the secondary structural elements from the crystal structure of ACRP30 on the alignment revealed that those residues forming β-strands of ACRP30 were mostly conserved in the NC1 domain, whereas differences were predominantly localized to the loop regions (Fig. 2). Therefore, the secondary structural elements are highly conserved. Within the β-strands, those residues forming the hydrophobic core of the monomer of ACRP30 (see below) are particularly conserved. This conservation strongly indicates that the tertiary fold of the NC1 domain is identical to the β-sandwich of ACRP30. In addition, the conservation of key interface residues between monomers (see below and Fig. 2) indicates that the quaternary structures of these two proteins are similar. This pattern of conservation, based on alignments, also holds across the complete family of related proteins including collagen types X and VIII, C1q, ACRP30, hibernation protein and cerebellin (see Fig. 1). A model of the trimeric NC1 domain (Fig. 3) was built using the three-dimensional coordinates of trimeric ACRP30 based on the alignment shown in Fig. 2. The amino-terminal 27 residues of the NC1 domain of type X collagen were not included in the model since there is no equivalent sequence in ACRP30 (Fig. 2). The 8 loop regions in the NC1 domain were modeled on the ACRP30 structure since their lengths are conserved, and they are anchored spatially by the β-strands. Upon trimerization (Fig. 3 a), the mode of packing between the NC1 monomer subunits produces a tight association contributed to by the loss of solvent-accessible surface and hence providing a free energy gain. The total buried solvent-accessible surface of the NC1 domain on trimerization is 6222 Å2, compared with 5324 Å2 for ACRP30. The three monomers pack together forming a twisted tunnel that extends through the core of the structure. The hydrophobic contacts that produce the close packing between the NC1 monomers are identical or show only conservative changes with respect to ACRP30. These include Ala553, Ile557, Leu575, Ile596, Val621, Ile641, Phe675, and Ala678 in the NC1 domain. However, in the NC1 domain additional residues (Pro550, Val551, Val668, and Pro679) contribute to hydrophobic interfaces between monomers. The equivalent residues in ACRP30 are Tyr114, Arg115, Asn233 and His244, respectively. Pro550, Val551, Ile641, Phe675, and Pro679 form a hydrophobic plug at the base of the NC1 trimer tunnel as viewed in Fig. 3 b. Moving up from the base, the trimer tunnel surface, contributed by all three monomers, becomes more polar with Tyr598, Ser600, Ser639, and Ser671 forming a dense network of hydrogen bonds with each other and water molecules (e.g. see Fig. 3 c). These polar residues are conserved across the entire family of type X collagen-related proteins (Fig. 1). Examining the monomer-monomer interfaces is complicated by the asymmetric association of the three monomers (designated M1—M3; see Fig. 3 a). Significant monomer-monomer contacts include a backbone hydrogen bond between the carbonyl oxygen of Tyr623 and the amide nitrogen of His669(between M2–M3 and M3–M1). M1–M2 has a different backbone contact involving the carbonyl oxygen of Gly631 and the amide nitrogen of Leu633. Examination of the NC1 trimer model revealed that the hydrophobic side chains of Pro568, Pro570, Ile588, Trp611, and Trp651 are exposed to solvent on the external surface of the trimer. All NC1 domain residues that are affected by single amino acid substitutions causing MCDS were displayed simultaneously on the monomer (Fig.3 d) and trimer (Fig. 3 e) models revealing a striking clustering of position. The substituted residues are localized to two distinct regions. The side chains of residues that are substituted in MCDS either form the polar and hydrogen-bonded region within the trimer tunnel (e.g. Tyr598 and Ser600, see Fig. 3 c) or form a patch on the external surface of the monomer/trimer assembled from part of loop D–E and part of β-strand G (Figs. 1 and 2). A detailed summary of the location and orientation of amino acid residues substituted within the NC1 domain in MCDS is presented in TableI.Table IPredicted location and orientation of NC1 domain amino acid substitutions causing MCDSPoint mutationPosition of residue and direction of side chainComments on wild type residue orientationY597HaIndicates residues located in a cluster on an interior patch.On β-strand C, interior face of trimer, side chain pointing to interior of monomerOH group hydrogen bonds to Ser552 via waterY598DaIndicates residues located in a cluster on an interior patch.On β-strand C, interior face of trimer, side chain pointing into trimer tunnelOH group forms pyramidal geometry with 2 other Tyr598 and a water molecule (see Fig. 3)S600PaIndicates residues located in a cluster on an interior patch.On β-strand C, interior face of trimer, side chain pointing into trimer tunnelOH groups from 3 monomers form a network of H bonds both with water and directly connecting the three monomers via Ser671 and Ser639. Proline could break the β-strandS671PaIndicates residues located in a cluster on an interior patch.On terminal β-strand H, on interior face of trimer with side chain pointing into monomer interfaceOne monomer forms a direct contact with neighboring monomer Ser600. Ser600 from M2 and M3 form a network of H bonds with water and both the side chain and backbone of neighboring Ser639. Proline could break the strandC591RbIndicates residues located on a surface patch on the exterior of trimer.On β-strand B on outside face of trimer with sulfydryl pointing to the interior of the monomerArginine could not be easily accommodated in the hydrophobic interiorG595EbIndicates residues located on a surface patch on the exterior of trimer.On bottom surface of trimer at the start of β-strand CThis glycine forms a sharp turn which would not be favorable for glutamic acidL614PbIndicates residues located on a surface patch on the exterior of trimer.On β-strand D, exterior face of trimer, side chain contributing to hydrophobic core of monomerThis strand immediately precedes loop DE (see Fig. 1). Proline could disrupt the β-strand conformation and dislocate the loopN617KbIndicates residues located on a surface patch on the exterior of trimer.On loop DE, exterior face of trimer, side chain points out into solutionThis loop maps onto corresponding loops in C1q and TNF-α which are involved in molecular recognitionG618VbIndicates residues located on a surface patch on the exterior of trimer.On loop DE, exterior face of trimer, this glycine forms the bend in the loopThis glycine has a φ torsion angle" @default.
• W2110231726 created "2016-06-24" @default.
• W2110231726 creator A5016572574 @default.
• W2110231726 creator A5022191527 @default.
• W2110231726 creator A5030068339 @default.
• W2110231726 creator A5031788018 @default.
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• W2110231726 date "1999-02-01" @default.
• W2110231726 modified "2023-10-17" @default.
• W2110231726 title "Metaphyseal Chondrodysplasia Type Schmid Mutations Are Predicted to Occur in Two Distinct Three-dimensional Clusters within Type X Collagen NC1 Domains That Retain the Ability to Trimerize" @default.
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