Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1

Nancy C M Caterina, L. Windsor, Audra E. Yermovsky, M. Kirby Bodden, Kenneth B. Taylor, Henning Birkedal-Hansen, Jeffrey A. Engler

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP- 1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity against M(r) 72,000 gelatinase (M(r) 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC → CQQC retained their ability to inhibit FIB-CL in a manner similar to wild- type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site 'parent' mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.

Original languageEnglish (US)
Pages (from-to)32141-32149
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number51
DOIs
StatePublished - Dec 19 1997
Externally publishedYes

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Tissue Inhibitor of Metalloproteinase-1
Cysteine
Matrix Metalloproteinase 8
Gelatinases
Substrates
Mental Competency
Peptides
Molecules
Aptitude
Vaccinia virus
Dichroism
Structural integrity
Gelatin
Circular Dichroism
human TIMP1 protein
Viruses
Human Activities
Disulfides
Trypsin
Serine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Caterina, N. C. M., Windsor, L., Yermovsky, A. E., Bodden, M. K., Taylor, K. B., Birkedal-Hansen, H., & Engler, J. A. (1997). Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1. Journal of Biological Chemistry, 272(51), 32141-32149. https://doi.org/10.1074/jbc.272.51.32141

Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1. / Caterina, Nancy C M; Windsor, L.; Yermovsky, Audra E.; Bodden, M. Kirby; Taylor, Kenneth B.; Birkedal-Hansen, Henning; Engler, Jeffrey A.

In: Journal of Biological Chemistry, Vol. 272, No. 51, 19.12.1997, p. 32141-32149.

Research output: Contribution to journalArticle

Caterina, NCM, Windsor, L, Yermovsky, AE, Bodden, MK, Taylor, KB, Birkedal-Hansen, H & Engler, JA 1997, 'Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1', Journal of Biological Chemistry, vol. 272, no. 51, pp. 32141-32149. https://doi.org/10.1074/jbc.272.51.32141
Caterina NCM, Windsor L, Yermovsky AE, Bodden MK, Taylor KB, Birkedal-Hansen H et al. Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1. Journal of Biological Chemistry. 1997 Dec 19;272(51):32141-32149. https://doi.org/10.1074/jbc.272.51.32141
Caterina, Nancy C M ; Windsor, L. ; Yermovsky, Audra E. ; Bodden, M. Kirby ; Taylor, Kenneth B. ; Birkedal-Hansen, Henning ; Engler, Jeffrey A. / Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 51. pp. 32141-32149.
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