Type I collagen cleavage is crucial for tissue remodeling, but its homotrimeric isoform is resistant to all collagenases. The homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of α1(I)3 homotrimers and normal α1(I)2α2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound α1(I) and α2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (⌬T m ) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of ⌬T m with the identity of the substituting residue. Instead, we observed regular variations in ⌬T m with the substitution location in different triple helix regions. To relate the ⌬T m map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (⌬G ‡ ) from the reported peptide data. We constructed the ⌬G ‡ map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the ⌬T m and ⌬G ‡ maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the ⌬T m map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the ␣1(I) chain.The mature type I collagen molecule is a 300-nm-long triple helix formed by two ␣1(I) and one ␣2(I) chains, which is flanked by short terminal peptides. Based on the (Gly-Xaa-Yaa) 338 triplet repeat within each chain (1), the triple helix is commonly viewed as a single domain (Xaa and Yaa stand for variable residues). However, this picture may not accurately represent variations in the stability of different regions within the triple helix that fold and unfold cooperatively (2-4). The triple helix is only metastable or marginally stable at physiological temperature (3, 5-8). Its local structure appears to be highly dynamic and intimately related to local stability. The more labile regions may exist in a loose conformation, constantly undergoing unfolding/refolding transitions while more stable "clamp" regions prevent unfolding of the whole molecule (3, 9 -12). Such structural and dynamic heterogeneity is believed to play an important role in self-assembly (9, 13) and function (3) of collagen fibers.Significant progress in understanding regional variations in the triple helix stability in different collagens has been reported in recent years. For example, some flexible sites were localized by observing triple helix bending in electron microscopy (14) and/or increased susceptibility to proteolytic cleavage (9, 15). Genetically generated reshuffling of different triple helix regions was shown to have a significant effect on the overall stability of the molecule (11, 16). Relative local stability maps were proposed based on scoring different sequences (17) or on the denaturation temperature (T m ) measured for triple-helical host-guest peptides (18).The existence of looser, less stable, and tighter, more stable structural regions within the triple helix is now commonly accepted. However, knowledge of their loca...

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