Abstract
The high resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) in solution has been determined using multidimensional heteronuclear NMR spectroscopy, with the structural calculations based on an extensive set of constraints, including 3132 nuclear Overhauser effect-based distance constraints, 56 hydrogen bond constraints, and 220 torsion angle constraints (an average of 26.9 constraints/residue). The core of the protein consists of a five-stranded beta-barrel that is homologous to the beta-barrel found in the oligosaccharide/oligonucleotide binding protein fold. The binding site for the catalytic domain of matrix metalloproteinases-3 (N-MMP-3) on N-TIMP-2 has been mapped by determining the changes in chemical shifts on complex formation for signals from the protein backbone (15N, 13C, and 1H). This approach identified a discrete N-MMP-3 binding site on N-TIMP-2 composed of the N terminus of the protein and the loops between beta-strands AB, CD, and EF. The beta-hairpin formed from strands A and B in N-TIMP-2 is significantly longer than the equivalent structure in TIMP-1, allowing it to make more extensive binding interactions with the MMP catalytic domain. A detailed comparison of the N-TIMP-2 structure with that of TIMP-1 bound to N-MMP-3 (Gomis-Ruth, F.-X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H. , Brew, K., Bourne, G. P., Bartunik, H. & Bode, W. (1997) Nature 389, 77-80) revealed that the core beta-barrels are very similar in topology but that the loop connecting beta-strands CD (P67-C72) would need to undergo a large conformational change for TIMP-2 to bind in a similar manner to TIMP-1.
Highlights
Remodeling of connective tissue is an important event in many normal and pathological processes such as growth, wound healing, tumor invasion, and rheumatoid and osteoarthritis
To obtain a high resolution solution structure for N-tissue inhibitors of metalloproteinases (TIMPs)-2, it was necessary to produce uniformly 15N- and 15N/13C-labeled N-TIMP-2. This was recently achieved by expression of the protein in Escherichia coli grown on labeled minimal medium followed by refolding of the labeled N-TIMP-2 from inclusion bodies [12]
High Resolution Solution Structure of N-TIMP-2—The solution structure of N-TIMP-2 was determined using a total of 3413 NMR-derived structural constraints (26.8/residue), including 3132 NOE-based upper distance limits (672 intraresidue, 784 sequential (i, i ϩ 1), 602 medium-range (i, i Յ 4), and 1074 long range (i, i Ն 5)), 56 hydrogen bonding constraints corresponding to 14 protein backbone hydrogen bonds and 220 torsion angle constraints (89 , 89 , and 42 1)
Summary
Remodeling of connective tissue is an important event in many normal and pathological processes such as growth, wound healing, tumor invasion, and rheumatoid and osteoarthritis. The binding site for the catalytic domain of matrix metalloproteinases-3 (N-MMP-3) on N-TIMP-2 has been mapped by determining the changes in chemical shifts on complex formation for signals from the protein backbone (15N, 13C, and 1H).
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