Title

Mutational study of the amino-terminal domain of human tissue inhibitor of metalloproteinases 1 (TIMP-1) locates an inhibitory region for matrix metalloproteinases

Date of Award

1997

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Keith Brew, Committee Chair

Abstract

TIMPs are protein inhibitors of the matrix metalloproteinases (MMPs), a family of enzymes that are responsible for the breakdown of components of connective tissue. Matrix turnover is an important aspect of many normal and pathological processes, such as growth, tissue remodeling, wound repair, and tumor metastasis.A bacterial expression system for the inhibitory N-terminal domain of the human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) has been established. N-TIMP-1 was expressed as inclusion bodies in E. coli and refolded to active protein in yields of twenty milligrams per liter of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the $\sp{15}$N-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis. The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2, and 3 is 6-8-fold less than that of the recombinant full-length protein, indicating that deletion of the C-terminal domain reduces the free energy of the protein-protein interaction by $<$10%.Seventeen single and double-site mutants have been produced using this expression system to probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in the K$\sb{\rm i}$ for stromelysin 1 (MMP-3) but do not disturb the three dimensional structure involve residues that are localized in a ridge that includes the N-terminal region of the protein. Specific residues that have a large influence on activity include Cys1, Thr2, Met66, Val69 and Cys70. Of the mutations introduced, the greatest functional disturbances, reflected in K$\sb{\rm i}$ increases of 2 to 3 orders of magnitude, are associated with the disruption of the Cys1-Cys70 disulfide bond or the substitution of Ala for Thr2. Most mutations that affect the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 and MMP-2, whereas the Thr2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that that it is possible to engineer TIMP-1 variants that are more specifically targeted to selected MMPs. The reactive site identified by these studies is a structurally constrained region of TIMP that has a suitable shape for fitting the MMP active site. Although its surface area is small in comparison with that expected for the magnitude of the MMP-TIMP interaction, adjacent regions with a strongly hydrophobic character may also contribute to the interaction interface.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9805944