title: Mutational and structural analyses of the hinge region of membrane type 1-matrix metalloproteinase and enzyme processing.
abstract: Membrane type 1 (MT1)-matrix metalloproteinase (MMP) is a major mediator of collagen degradation in the pericellular space in both physiological and pathological conditions. Previous evidence has shown that on the cell surface, active MT1-MMP undergoes autocatalytic processing to a major membrane-tethered 44-kDa product lacking the catalytic domain and displaying Gly285 at its N terminus, which is at the beginning of the hinge domain. However, the importance of this site and the hinge region in MT1-MMP processing is unknown. In the current study, we generated mutations and deletions in the hinge of MT1-MMP and followed their effect on processing. These studies established Gly284-Gly285 as the main cleavage site involved in the formation of the 44-kDa species. However, alterations at this site did not prevent processing. Instead, they forced downstream cleavages within the stretch of residues flanked by Gln296 and Ser304 in the hinge region, as determined by the processing profile of various hinge deletion mutants. Also, replacement of the hinge of MT1-MMP with the longer MT3-MMP hinge did not prevent processing of MT1-MMP. Molecular dynamic studies using a computational model of MT1-MMP revealed that the hinge region is a highly motile element that undergoes significant motion in the highly exposed loop formed by Pro295-Arg302 consistent with being a prime target for proteolysis, in agreement with the mutational data. These studies suggest that the hinge of MT1-MMP evolved to facilitate processing, a promiscuous but compulsory event in the destiny of MT1-MMP, which may play a key role in the control of pericellular proteolysis.
authors.affiliation: Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Pamela
authors.initials: P
authors.lastname: Osenkowski
authors.forename: Samy O
authors.initials: SO
authors.lastname: Meroueh
authors.forename: Dumitru
authors.initials: D
authors.lastname: Pavel
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.forename: Rafael
authors.initials: R
authors.lastname: Fridman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D002384:Catalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D000882:Haplorhini; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D053513:Matrix Metalloproteinase 16; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014616:Vaccinia virus / Q000378:metabolism
chemical_list: C507102:MMP16 protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012694:Serine; D001120:Arginine; D011392:Proline; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D005998:Glycine