Structure-function relationships in human tissue inhibitor on metalloproteinase (TIMP)
Date of Award
Doctor of Philosophy (Ph.D.)
Biochemistry and Molecular Biology
First Committee Member
Keith Brew, Committee Chair
A computer model of N-TIMP-2/MMP-1 (fibroblast collagenase) complex was generated based on previous mutational studies in which a surface ridge was identified as being a key to the inhibitory action, and this inhibitory region is composed from two stretches of sequences that are linked by the Cys 1-Cys 70 disulfide bond. Mutants of N-TIMP-1 were generated to test the hypotheses regarding the action of this region based on computer modeling. The importance of the alpha-NH2 group of Cys 1 in interaction with the catalytic site, Thr 2 in interacting with the specificity pocket and a hydrophobic patch in the TIMP-2/MMP-1 model were tested. An N-terminal extension of one residue (-1A) reduced affinity for MMP-3 250-fold, while substitutions for Phe 101 and Val 102 disrupt both activity and structure. Both the computer model of the N-TIMP-2/MMP-1 complex and recently determined crystal structure of a TIMP-1/MMP-3 (stromelysin) complex indicate that the side chain of residue 2 of TIMP fits into the S1' pocket of the metalloproteinase and the alpha-amino and carbonyl groups of Cys 1 act in coordinating the catalytic zinc in MMP active site. Substitutions of components of an apparently exposed hydrophobic (Phe 103- Pro 106) observed in the TIMP-2 model were found to affect structure, in agreement with its buried state in the TIMP-1 crystal structure.Twelve mutants at position 2 (Thr 2 Ser, Met, Leu, Ile, Phe, Gly, Asp, Arg, Glu, Lys, Gln, His) were designed to probe roles of different features of side chain chemistry in binding to MMP-1, -2 and -3. Results show that (1) the presence of side chain at position 2 is necessary, as elimination of this side chain by mutagenesis (Thr 2 to Gly) generates TIMP with very weak affinities for MMPs. (2) A long hydrophobic side chain at position 2 is more favorable than charged side chains for binding to all three MMPs. T2M is the best inhibitor for MMP-2 and MMP-3 among all 12 mutants, having higher binding affinities compared with the parental N-TIMP-1. (3) Some N-TIMP-1 variants, particularly, T2L, T2I, T2R, T2K and T2Q, have a strong selectivity against MMP-1. (4) Smaller side chain (T2S, T2G and T2A) are more favorable for binding to MMP-3 than MMP-2, and -1. Mutations with altered inhibitory properties were examined by CD spectroscopy to determine if the substitution could affect activity by inducing a significant change in structure. All the Thr 2 mutants have very similar CD spectrum to wild type N- TIMP-1.In summary, position 2 is a key residue for TIMP specificity and function in keeping with the dominant role of the P1' residue of peptide substrates in MMP specificity. However the effects of different residues at position 2 of N-TIMP-1 on MMP binding do not parallel the effects of the substrate P1' residue indicating a different mode of binding for this residue in the TIMP/MMP complex. T2 mutants have altered specificity against different MMPs, particularly MMP-1. However, multiple site substitutions in the inhibitory region may be necessary to generate highly specific inhibitors.
Biology, Molecular; Chemistry, Biochemistry
Meng, Qi, "Structure-function relationships in human tissue inhibitor on metalloproteinase (TIMP)" (1998). Dissertations from ProQuest. 3565.