Sergei P. Boudko

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon.

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.

Crystal structure of human type III collagen Gly991-Gly1032 cystine knot-containing peptide shows both 7/2 and 10/3 triple helical symmetries.

Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3Å resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.

Mutation in cyclophilin B that causes hyperelastosis cutis in american quarter horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin b-protein interactions and affects collagen folding

Background: Hyperelastosis cutis in horses is caused by a homozygous mutation in cyclophilin B (PPIB). Results: This mutation changes protein-protein interactions of CypB and delays folding of collagen. Conclusion: Mutant CypB has PPIase activity, but the synthesized collagen contains less post-translational modifications of lysine residues. Significance: CypB has other important functions besides its PPIase activity. The formation of these interactions is crucial for a correct biosynthesis of collagen. The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.

Crystal structure of human collagen XVIII trimerization domain: A novel collagen trimerization Fold.

Collagens contain a unique triple-helical structure with a repeating sequence -G-X-Y-, where proline and hydroxyproline are major constituents in X and Y positions, respectively. Folding of the collagen triple helix requires trimerization domains. Once trimerized, collagen chains are correctly aligned and the folding of the triple helix proceeds in a zipper-like fashion. Here we report the isolation, characterization, and crystal structure of the trimerization domain of human type XVIII collagen, a member of the multiplexin family. This domain differs from all other known trimerization domains in other collagens and exhibits a high trimerization potential at picomolar concentrations. Strong chain association and high specificity of binding are needed for multiplexins, which are present at very low levels.

Cole-Carpenter Syndrome Is Caused by a Heterozygous Missense Mutation in P4HB

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.

The crucial role of trimerization domains in collagen folding

Collagens contain large numbers of Gly-Xaa-Yaa peptide repeats that form the characteristic triple helix, where the individual chains fold into a polyproline II helix and three of these helices form a right-handed triple helix. For the proper folding of the triple helix collagens contain trimerization domains. These domains ensure a single starting point for triple helix formation and are also responsible for the chain selection in heterotrimeric collagens. Trimerization domains are non-collagenous domains of very different structures. The size of trimerization domains varies from 35 residues in type IX collagen to around 250 residues for the fibrillar collagens. These domains are not only crucial for biological functions, but they are also attractive tools for generating recombinant collagen fragments of interest as well as for general use in protein engineering and biomaterial design. Here we review the current knowledge of the structure and function of these trimerization domains.

Kinetic Hysteresis in Collagen Folding

The triple helix of collagen shows a steep unfolding transition upon heating, whereas less steep and more gradual refolding is observed upon cooling. The shape of the hysteresis loop depends on the rate of temperature change as well as the peptide concentration. Experimental heating and cooling rates are usually much faster than rates of unfolding and refolding. In this work, collagen model peptides were used to study hysteresis quantitatively. Their unfolding and refolding profiles were recorded at different heating and cooling rates, and at different peptide concentrations. Data were fitted assuming kinetic mechanisms in which three chains combine to a helix with or without an intermediate that acts as a nucleus. A quantitative fit was achieved with the same kinetic model for the forward and backward reactions. Transitions of exogenously trimerized collagen models were also analyzed with a simplified kinetic mechanism. It follows that true equilibrium transitions can only be measured at high concentrations of polypeptide chains with slow scanning rates, for example, 0.1 degrees C/h at 0.25 mM peptide concentration of (Gly-Pro-Pro)(10). (Gly-Pro-4(R)Hyp)(10) folds approximately 2000 times faster than (Gly-Pro-Pro)(10). This was explained by a more stable nucleus, whereas the rate of propagation was almost equal. The analysis presented here can be used to derive kinetic and thermodynamic data for collagenous and other systems with kinetically controlled hysteresis.

Collagen formation and structure

This chapter reviews the current knowledge of collagens, a protein family important for the extracellular matrix of vertebrate animals. The 28 types of collagens are discussed in terms of their structure, their distribution in tissues, and their involvement in human diseases. The biosynthesis of collagens with their numerous posttranslational modifications is reviewed. The current views on chain selection, trimerization, and folding of collagens and collagen-like peptides are described and recent advances in the determination of the atomic structure of the triple helix are also discussed.

Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain

The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization.