Structure and function of MMP-2 and its inhibitor TIMP-2

Abstract

Animal cells secrete various enzymes capable of degrading the extracellular matrix. One of the most studied groups of these are the matrix metalloproteinases (MMPs). This group of zinc containing endopeptidases consists at present of 26 members and this number is still increasing.

MMPs have partially overlapping specificity for various extracellular matrix substrates. Lately this specificity has extended to proteins outside the matrix surrounding cells and organs. MMPs are linked to various pathological functions such as cancer metastasis, arthritis and angiogenesis, as well as physiological functions like wound healing, bone reformation, involution etc.

Matrix metalloproteinase 2 (MMP-2, gelatinase A, 72 kDa type IV collagenase) degrades many extracellular matrix proteins, and it is known to specifically cleave type IV collagen, the major component of the basement membranes. Tissue inhibitors of metalloproteinases (TIMPs) are specific protein inhibitors for matrix metalloproteinases. Thus MMP-2 and TIMPs are key proteins for processes where cells move through basement membranes, for example during development, immune defense, but also in angiogenesis and tumor metastasis.

TIMP-2 and MMP-2 were expressed as recombinant proteins, for studying biochemical properties and to allow crystallization and the determination of the 3D structure.

The 3D structure of human proMMP-2 is the first full-length structure of any metalloproteinase from any species. The protein was crystallized as a proteolytically inactive mutant to avoid degradation during crystallization. The propeptide domain binds to the catalytic zinc through the cysteine residue in the conserved PRCGVPD sequence to the enzyme in an inactive proform (cysteine-switch mechanism). The catalytic domain is structurally highly homologous to other known MMP structures, except that it contains three additional fibronectin type II domains responsible for substrate specificity. The C- terminal domain of MMP-2 (hemopexin like domain) is a four-bladed propeller. The structure contributes significally to our understanding of the activation mechanism of metalloproteinases. However, substrate binding and recognition remains obscure. The orientation of fibronectin type II domains was found to be in contradiction with models derived from theoretical predictions.

The structure of TIMP-2 is very closely related to the previously known structure of TIMP-1. The amino terminal domain folds into an 013-fold, which is found in many oligosacharide-oligonucleotide binding proteins. This [beta]-barrel domain contains the inhibitory activity of this protein and binds with its N-terminal residue in a bidentate manner to the catalytic zinc of the metalloproteinases.