The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2).
The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T(1) and T(2) relaxation times and (1)H-(15)N nuclear Overhauser effect values for the backbone amide (15)N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended beta-hairpin between beta-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2. N-MMP-3 complex, the rapid internal motion of the AB beta-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB beta-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.
Williamson
R A
RA
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom.
Muskett
F W
FW
Howard
M J
MJ
Freedman
R B
RB
Carr
M D
MD
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry* / Q000378:metabolism
D019716:Tissue Inhibitor of Metalloproteinase-2; D019278:Matrix Metalloproteinase 3