title: The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.
abstract: Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.
authors.affiliation: From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Lauer
authors.forename: Manishabrata
authors.initials: M
authors.lastname: Bhowmick
authors.forename: Dorota
authors.initials: D
authors.lastname: Tokmina-Roszyk
authors.forename: Yan
authors.initials: Y
authors.lastname: Lin
authors.forename: Steven R
authors.initials: SR
authors.lastname: Van Doren
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D020816:Amino Acid Motifs; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D055672:Static Electricity
chemical_list: D003094:Collagen; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: Charge Clusters; Collagen; Collagenolysis Mechanism; Extracellular Matrix Proteins; Matrix Metalloproteinase (MMP); Protease; Protein Folding; Triple Helix