Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition

Maryam Raeeszadeh-Sarmazdeh,Kerrie A Greene,Banumathi Sankaran,Gregory P Downey,Derek C Radisky,Evette S Radisky

doi:10.1074/jbc.ra119.008321

Abstract

Tissue 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 ultrabinders. 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 WT 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.

Highlights

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases
The free, exposed N terminus of TIMP-1 is required for MMP binding and inhibition [6, 23]; we designed a fusion construct connecting the C terminus of TIMP-1 to the N terminus of yeast cell wall protein Aga2p, with secretion directed by the yeast ␣-factor signal sequence (Fig. 1A)
The optimized construct was N-terminally processed by yeast protease Kex2, resulting in efficient display of a functional TIMP-1 fusion that demonstrated robust MMP binding, as detected using biotinylated MMP-3 catalytic domain (MMP-3cd) (Figs. 1C and 2, A and D)

Summary

Introduction

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. 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. 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. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The TIMP C-terminal domain may contribute to the binding interface with an MMP catalytic domain, conferring a degree of binding affinity [11, 12], the significance of this interaction and mechanism by which it contributes to MMP recognition have been largely overlooked. We sought to probe the role of the TIMP C-terminal domain and of cooperativity between TIMP domains in MMP recognition

Objectives

Results

Conclusion