Characterization of the Distinct Collagen Binding, Helicase and Cleavage Mechanisms of Matrix Metalloproteinase 2 and 14 (Gelatinase A and MT1-MMP)

Abstract

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of ␣-chain hydrolysis.

pH dependence of the enzymatic processing of collagen I by MMP-1 (fibroblast collagenase), MMP-2 (gelatinase A), and MMP-14 ectodomain

“…The triple-helix nature of collagen was checked by circular dichroism (CD) measurements and further confirmed by the trypsin degradation assay. This assay validates the quality of collagen triple-helix structure, verifying the absence of collagen degradation at 1:10 trypsin to collagen ratio over 3 h at 28°C, as described previously [11]. Lyophilized collagen was stored at -80°C, and stock solutions were prepared as needed.…”

“…MMPs are virtually capable of proteolytically processing all molecules present in the ECM, though they display differing propensity for various substrates [1][2][3]. From the structural viewpoint, all MMPs share a common catalytic site, where a Zn 2? ion is coordinated by three histidyl residues, whereas they differ regarding the presence and the structural arrangement of additional domains, such as the hemopexin-like domain and the fibronectin-like domain, which are important for the recognition of macromolecular substrates [9][10][11][12][13].…”

“…On the basis of their structural features and of their propensity to proteolytically process collagen I, MMPs are classified into five main groups, namely, (1) collagenases (i.e., MMP-1, MMP-8, and MMP-13), which preferentially cleave collagen I, recognizing the substrate through the hemopexin-like domain [10,17,18], (2) gelatinases (i.e., MMP-2 and MMP-9), which are able to cleave both collagen I and collagen IV [19,20], recognizing the substrates preferentially through the fibronectin-like domain [9,11,13,20], a fibronectin II-like domain inserted only in the catalytic domain of gelatinases, (3) stromelysins (i.e., MMP-3, MMP-10, and MMP-11), which enzymatically process collagen IV, but not collagen I [3], (4) matrilysins (i.e., MMP-7 and MMP-26), which are unable to cleave collagen I [21,22], and (5) membrane-type MMPs (i.e., MMP-14, MMP-15, MMP-16, MMP-17, and MMP- 24), which display at the C-terminal a transmembrane domain with a short cytoplasmic tail and a long extracellular ectodomain; among these, only MMP-14 is able to enzymatically process collagen I [23]. Hence, only collagenases, gelatinases, and MMP-14 are able to cleave collagen I.…”

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

“…The function of the CBD of MMPs in collagenolysis has been intensively studied. Most studies show that MMPs unwind triple-helical collagen prior to peptide bond hydrolysis because the catalytic sites of MMPs are too narrow to accommodate the triple-helical structure of collagen (16,26,27). The CBD of MMPs has triple-helicase activity and induces local structural disruption of the collagen triple helix.…”

“…The CBD of MMPs has triple-helicase activity and induces local structural disruption of the collagen triple helix. Then the catalytic domain of MMPs cleaves the three chains one by one (27). Another hypothesis for the action of MMP CBDs on triple-helical collagen posits that the binding of the CBD can stabilize partially unfolded conformers so that the catalytic domain can recognize the unwound part and initiate collagen degradation (28).…”