Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1

Darin L. Steele, O. El-Kabbani, P. Dunten, L. Jack Windsor, R. Ursula Kammlott, R. L. Crowther, C. Michoud, Jeffrey A. Engler, J. J. Birktoft

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) family of enzymes. The active site glutamic acid of the MMPs is conserved throughout the family and plays a pivotal role in the catalytic mechanism. The structural and functional consequences of a glutamate to glutamine substitution in the active site of stromelysin-1 were investigated in this study. In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activated by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue inhibitor of metalloproteinases, after cleavage of the propeptide with trypsin, A second construct containing the glutamine substitution but lacking the propeptide was also inactive in the proteolysis assays and capable of TIMP-1 binding. X-ray structures of the wild-type and mutant proteins complexed with the propeptide-based inhibitor Ro-26-2812 were solved and in both structures the inhibitor binds in an orientation the reverse of that of the propeptide in the pro-form of the enzyme. The inhibitor makes no specific interactions with the active site glutamate and a comparison of the wildtype and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.

Original languageEnglish (US)
Pages (from-to)397-405
Number of pages9
JournalProtein Engineering
Volume13
Issue number6
DOIs
StatePublished - Jan 1 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Steele, D. L., El-Kabbani, O., Dunten, P., Windsor, L. J., Kammlott, R. U., Crowther, R. L., Michoud, C., Engler, J. A., & Birktoft, J. J. (2000). Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1. Protein Engineering, 13(6), 397-405. https://doi.org/10.1093/protein/13.6.397