Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases*

Abstract

The mechanism by which enzymes recognize the "uniform" collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Glyϳ[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P 3 subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.The degradation of collagen, the major structural component of connective tissues in skin, bone, tendon, and ligament, is an integral part in many biological processes such as wound healing, cell migration, tissue remodeling, and organ morphogenesis (1-4). Accelerated breakdown of collagen may result in many diseases such as arthritis, tumor cell invasion, glomerulonephritis, and metastasis (5-7). Types I, II, and III collagens, also called interstitial collagens, are the most abundant (8 -10), and contain a characteristic triple-helical conformation, which consists of three polyproline II-like helices supercoiled around a common axis (11,12). The close packing of the three chains can only accommodate Gly as every third residue, generating the repetitive (Gly-Xaa-Yaa) n sequence pattern. The Gly residues are all buried in the center, and the structure is stabilized by interchain N-H (Gly) . . . CϭO (Xaa) hydrogen bonds. The residues at the X and Y positions can be almost any amino acid, but they are frequently Pro and 4-hydroxyproline (Hyp 2 or O), respectively.The triple helical structure allows collagen to be degraded by only a few proteinases including a group of matrix metalloproteinases (MMPs) (5, 13). These MMPs (MMP-1, MMP-2, MMP-8, MMP-13, MMP-14, MMP-18) can bind and cleave interstitial collagens at a unique locus approximately threefourths away from the N terminus of the collagens (14). The cleavage site is after the Gly residue in the sequence of Glyϳ[Ile/Leu]-[Ala/Leu]...

Interstitial Collagen Catabolism

“…The Ile residue in one of the three chains at the site of MMP hydrolysis has a distinct chemical shift, a higher J coupling value, increased dynamics, and decreased local stability (49). This suggests that a single locally dynamic chain, rather than a labile region with three comparably dynamic chains, is a determining factor for collagen to be cleaved by MMPs (49).…”

“…This suggests that a single locally dynamic chain, rather than a labile region with three comparably dynamic chains, is a determining factor for collagen to be cleaved by MMPs (49). Also, a Pro residue at the P 3 subsite influences the P 1 Ј subsite Ile residue, enhancing its accessibility to collagenolytic MMPs (49).…”

Flow cytometry in cancer stem cell analysis and separation

“…As an alternative to trypsin, tissue dissociation can be achieved using a mixture of hyaluronidase, DNAse, and collagenases (92). Although this preparation also contains a proteolytic enzyme, collagenase mainly digests extracellular matrix molecules containing unique structural motifs not commonly present in most cell surface epitopes relevant to the identification or sorting of CSCs (93). With respect to cultured primary tumor cells and tumor cell lines which contain subpopulations of CSCs (22,63,66), a detachment by the Ca 21 -chelating agent EDTA can serve as a viable alternative to trypsinization.…”

The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite

“…In fibrillar collagens, the region surrounding the collagenase cleavage site contains numerous nonpolar residues (which are otherwise rare) and has a low imino acid content that together may make the local triple helical conformation relatively unstable at physiological temperatures (15,16). Furthermore, Ile 776 in the leading strand is more conformationally labile than the equivalent residue in the middle and lagging strands, and thus may serve as a signal for collagenase cleavage (17). In turn, the collagenolytic MMPs contain numerous "exosites," regions that, although lacking in any hydrolytic apparatus (this is limited to the active site of the CAT domain), are required for recognition and cleavage of the natural substrate (reviewed by Ref.…”