Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate.
The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.
Meng
Q
Q
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Malinovskii
V
V
Huang
W
W
Hu
Y
Y
Chung
L
L
Nagase
H
H
Bode
W
W
Maskos
K
K
Brew
K
K
D002460:Cell Line; D002942:Circular Dichroism; D017364:Collagenases / Q000378:metabolism; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D007700:Kinetics; D020781:Matrix Metalloproteinase 1; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000378:metabolism; D015854:Up-Regulation
D017931:DNA Primers; D019715:Tissue Inhibitor of Metalloproteinase-1; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1