Collagen folding and Interactions: from basic principles to bone disorders

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Type I collagen is a long, triple helical protein, which forms the matrix of bone, skin and other tissues. During the last year, we continued characterization of structural consequences of Osteogenesis Imperfecta (OI) mutations in the type I triple helix. We constructed the map of changes in the triple helix stability (melting temperature) caused by 36 point mutations at 34 different sites along the helix. To relate this map to peptide-based stability predictions, we proposed a model for extracting the activation energy of local helix unfolding from the reported peptide data. We tested the model and determined its parameters by measuring the H-D exchange rate for glycine NH residues involved in inter-chain hydrogen bonds in collagen. From comparison of the peptide-based and mutation-based stability maps, we refined our model of regional variations in the triple helix structure. The refined regions align with regions important for collagen fibril assembly and ligand binding, and they appear to contribute to at least some of the observed regional variations in OI phenotype. Disruption of collagen interactions with ligands, particularly other extracellular matrix proteins and proteoglycans, is one of possible mechanisms relating structural defects in collagen triple helix to functional abnormalities in OI. Therefore, we are developing a novel confocal microscopy assay, which will allow one to visualize and quantitatively measure binding of various matrix proteins to individual collagen fibrils based on differential fluorescent labeling of collagen and the ligand. With this assay, e.g., we found that mammalian collagenases attack damaged and poorly assembled collagen fibers via preferential binding to microfibrils, which become more exposed at fiber defects. In the last year, we focused on competitive binding experiments between fluorescently labeled and unlabeled decorin, which revealed binding enhancement dependent on the number of fluorescent labels attached to decorin. Based on these measurement, we developed a protocol for characterization and correction for such fluorophore effects. Presently, we are conducting systematic measurements of interactions between collagen fibers and different matrix proteins potentially important in OI and other connective disuse disorders. The overwhelming majority of severe OI cases are caused by single amino acid substitutions. However, several recessive OI and EDS cases were described, in which all type I collagen was synthesized in the form of alpha-1 homotrimer rather than the normal heterotrimer of two alpha-1 and one alpha-2 chains. Formation of type I homotrimers was also observed in cultures of breast cancer cells and in cultures of normal bone cells from individuals predisposed to common, age-related osteoporosis. Our previous studies revealed altered regional stability of the homotrimer triple helix and a small increase in the denaturation temperature of the whole molecule, but they did not offer clues to potential mechanisms of pathology. During the last year, we made a potential breakthrough. We discovered a significant reduction in the homotrimer cleavage rate by major tissue collagenases, MMP-1 and MMP-13. More detailed examination suggested that the initial MMP binding to collagen is not affected by the lack of the alpha-2 chain, but that this chain is essential for the next step of triple helix unwinding and opening, preceding the cleavage. In tissues with mixed collagen composition, e.g., containing type I homo- and heterotrimers or type I homotrimers and type III collagen, the abnormal cleavage rate of one component will alter the normal remodeling process. We believe that the resulting abnormal remodeling may play an important role in various pathologies associated with homotrimer synthesis and hope that better understanding of the underlying molecular mechanism may help to develop new treatment strategies. Most but not all cases with clinical symptoms of OI are caused by mutations in type I collagen. In collaboration with clinical researchers from Bone and Extracellular Matrix Branch of NICHD, we just reported that severe/lethal skeletal pathology reminiscent of OI is also caused by recessive null mutations in the cartilage associated protein (CRTAP) or prolyl-3-hydroxylase (P3H1). CRTAP and P3H1 form a tight three-protein complex with cyclophilin B in the Endoplasmic Reticulum (ER). Our measurements revealed that the disruption of this complex significantly delays type I procollagen folding, resulting in overhydroxylation and overglycosylation of Lys residues, which is also observed in many OI cases. We believe that the CRTAP and P3H1 may be essential for retaining cyclophilin B within ER and that the prolyl isomerase activity of cyclophilin B is essential for procollagen folding. This hypothesis is currently under investigation in several research groups.