6mav
Complex of tissue inhibitor of metalloproteinase-1 (TIMP-1) mutant L34G with matrix metalloproteinase-3 catalytic domain (MMP-3cd)Complex of tissue inhibitor of metalloproteinase-1 (TIMP-1) mutant L34G with matrix metalloproteinase-3 catalytic domain (MMP-3cd)
Structural highlights
DiseaseMMP3_HUMAN Defects in MMP3 are the cause of susceptibility to coronary heart disease type 6 (CHDS6) [MIM:614466. A multifactorial disease characterized by an imbalance between myocardial functional requirements and the capacity of the coronary vessels to supply sufficient blood flow. Decreased capacity of the coronary vessels is often associated with thickening and loss of elasticity of the coronary arteries. Note=A polymorphism in the MMP3 promoter region is associated with the risk of coronary heart disease and myocardial infarction, due to lower MMP3 proteolytic activity and higher extracellular matrix deposition in atherosclerotic lesions.[1] [2] FunctionMMP3_HUMAN Can degrade fibronectin, laminin, gelatins of type I, III, IV, and V; collagens III, IV, X, and IX, and cartilage proteoglycans. Activates procollagenase. Publication Abstract from PubMedTissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultra-binders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with wild-type TIMP-1, with inhibition constants (Ki) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface, and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems. Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition.,Raeeszadeh-Sarmazdeh M, Greene KA, Sankaran B, Downey GP, Radisky DC, Radisky ES J Biol Chem. 2019 Apr 30. pii: RA119.008321. doi: 10.1074/jbc.RA119.008321. PMID:31040180[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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