Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases

Abstract

Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum. We observe that the application of 16 pN of force causes an 8-fold increase in collagen proteolysis rates by MMP-1 but does not affect cleavage rates by Clostridium collagenase. Quantitative analysis of these data allows us to infer the structural changes in collagen associated with proteolytic cleavage by both enzymes. Our data support a model in which MMP-1 cuts a transient, stretched conformation of its recognition site. In contrast, our findings suggest that Clostridium collagenase is able to cleave the fully wound collagen triple helix, accounting for its lack of force sensitivity and low sequence specificity. We observe that the cleavage of heterotrimeric collagen is less force sensitive than the proteolysis of a homotrimeric collagen model peptide, consistent with studies suggesting that the MMP-1 recognition site in heterotrimeric collagen I is partially unwound at equilibrium.

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

“…Such a mechanochemical switch has been observed by Flynn et al, who reported that strain inhibited degradation of tissue-derived fibrils by bacterial collagenase (16) and reconstituted fibrils by MMP-8 (15). There are conflicting reports on the effect of tension on enzymatic cleavage of isolated triple helices (42)(43)(44); however, these measurements are of uncertain significance with regard to natively assembled fibrils or collagen structures in tissues. Decisively, tension-dependent stabilization against MMP-1 has been demonstrated in native tissue (17).…”

Genetically modified human type II collagen for N- and C-terminal covalent tagging

“…[26] Aldehydes have previously been introduced into the N-terminus of collagens via reaction with the small molecule pyridoxal 5′-phosphate (PLP). [14,17] We propose an alternative strategy, which produces aldehydes endogenously. This would facilitate tagging in cell-based experiments.…”

“…[39] If yeast-expressed collagen, produced with only prolyl hydroxylation as a post-translational modification, is sufficient for experiments, then aldehyde modification via the PLP small-molecule reaction offers easy access to labelled collagen. [14,17] However, if collagen harboring a more complete suite of modifications is desired, a different cell line must be used. [38] Based on the findings of this work, we propose that inhibition [34] or a knockout of lysyl oxidase in a human-cell-based recombinant expression system could result in the ability to introduce aldehydes in collagen uniquely located within the FGE recognition sequence and available for specific chemical tagging.…”

“…[14] This approach may have low efficacy due to the partial removal of telopeptides in the pepsin digestion used to produce the collagen. Additionally, antibody linkages are mechanically weaker than other linking strategies, such as biotin-streptavidin or covalent interactions.…”

“…The latter have been used to address one end of type I collagen or the two distinct ends of type III collagen, but in both cases these have used commercially available protein, which is not amenable to sequence manipulation for downstream structure-function studies. [14,17] There is a need for specific functionalization of collagen produced from a recombinant expression system, which would facilitate studies of how mutations disrupt the triple helix. This ability to chemically address collagen would enable detailed single-molecule mechanical studies, ideally to high forces, and also be applicable to fluorescence imaging via coupling to fluorophores or other labels of interest.…”