The Intermediate S1′ Pocket of the Endometase/Matrilysin-2 Active Site Revealed by Enzyme Inhibition Kinetic Studies, Protein Sequence Analyses, and Homology Modeling

Abstract

Matrix metalloproteinases (MMPs, 1 matrixins) are believed to participate in angiogenesis, embryonic development, morphogenesis, reproduction, tissue resorption and remodeling, and tumor growth, progression, invasion, and metastasis through breakdown of the extracellular matrix, cell surface proteins, and processing growth factors, cytokines, and chemokines (1-3). Recently, human MMP-26 (endometase/matrilysin 2) was identified and its mRNA expression was detected in normal tissues of the human uterus and placenta, and in many types of malignant tumors (4 -7). Characterization of the MMP-26 promoter suggests that this proteinase may be expressed in cancer cells of epithelial origin (8). MMP-26 may play an important role in human prostate and breast cancer invasion (9 -10).MMP-26 cleaves type I gelatin, ␣ 1 -proteinase inhibitor, fibrinogen, fibronectin, vitronectin, type IV collagen, and insulin-like growth factor binding protein-1 (4, 7, 11). Studies of MMP-26 indicate that it has substrate specificity similar to other MMPs, with the exception of a preference for Ile at the P 2 and P 2 Ј positions, for small residues at the P 3 Ј and P 4 Ј positions, and Lys at the P 4 position (11). MMP-26 also hydrolyzes several synthetic fluorogenic peptide substrates designed for stromelysin-1, gelatinases, collagenases, and tumor necrosis factor-␣ converting enzyme (4, 11). According to these peptide substrate studies, MMP-26 may be capable of cleaving a broad range of substrates, although it has less catalytic efficiency than other MMPs.X-ray crystal structures of MMPs illustrate that overall topology and secondary structures are conserved (12-18). The S 1 Ј pocket, a hydrophobic pocket of variable depth, is a well defined substrate P 1 Ј-binding site in MMPs. Three types of S 1 Ј pockets can be distinguished from the available structures of . One type is a shallow pocket, as found in MMP-1 (human fibroblast collagenase; 13) and 16), where the pockets are limited by the side chains of Arg and Tyr, respectively, crossing the pockets. Many of the structurally known MMPs possess Leu at the corresponding site, and its side chain forms the top of the pocket rather than crossing the pocket. These Leu-containing MMPs may be further classified as deep and intermediate S 1 Ј pocket MMPs. A deep, tunnel-like pocket is found in MMP-3 (stromelysin-1; 12), MMP-12 (metalloelastase; 17), and MMP-14 (MT1-MMP; 21), whereas MMP-2 (gelatinase A; 22), MMP-8 (human neutrophil collagenase; 15), and MMP-9 (gelatinase B; 23) possess an intermedi-

Expression of matrix metalloproteinase-26 promotes human glioma U251 cell invasion in vitro and in vivo

“…Published research has shown that MMPs can promote tumor invasion through tissue barriers by degrading extracellular matrix and disrupting the basement membrane (36). MMP-26 can degrade most of the ECM components including fibronectin, vitronectin, fibrinogen, type IV collagen, gelatin, and non-ECM proteins including ·1-proteinase inhibitor and growth factor-binding protein 1 in vitro (11)(12)(13). Our study showed that MMP-26 expression contributes to the adherence and spreading ability of U251 cells on extracellular matrix components which contributes to the invasion process in vitro.…”

“…This special structural feature is to some extent a reflection of its unconventional enzyme activation mechanism, transcriptional regulation mechanism, and unusual function in physiological and pathological processes. MMP-26 can degrade most of the ECM components (11)(12)(13). MMP-26 can also activate proMMP-9 through cleavage of Ala93-Met94 site of the prepro-enzyme (14).…”

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

“…We used the same sequencing numbering as given by Park et al [4]. A homology modelling structure of MMP-26 was constructed based on the crystal structure of the catalytic domain of MMP-12 (cdMMP-12) [47]. A comparison of the MMP-26 structure with cdMMP-12 predicted three putative calcium-binding sites, designated C1, C2 and C3 in Figure 1(A) Wild-type pro-MMP-26 was auto-activated during folding by dialysis [4,8,40].…”

“…The homology modelling structure of the MMP-26 catalytic domain was constructed previously [47] using the SwissModel program [48]. The crystal structure of MMP-12 [33], which exhibits high similarity to MMP-26, was used as a template (PDB accession code 1JK3).…”

“…The crystal structure of MMP-12 [33], which exhibits high similarity to MMP-26, was used as a template (PDB accession code 1JK3). Minimization calculations were performed with an Amber forcefield modified to include parameters for zinc and calcium [47]. Both of the putative low-affinity Ca 2+ ions at the C2 and C3 sites were removed, and the protein structure was minimized to examine the resultant structural changes.…”