Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Wenting Bu,Suet-Mien Tan,Meitian Wang,Yong-Gui Gao,Xinfu Yan,So Fong Cam Ngan,Zhi Yang Loh,Zarina Levitskaya,Siu Kwan Sze,Shibom Basu,Shengyang Jin,Rya Ero,Raquel L Lieberman

doi:10.1371/journal.pbio.3000755

Abstract

Kindlin-1, -2, and -3 directly bind integrin β cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin β cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

Highlights

Integrin-mediated cell–cell and cell–extracellular matrix interactions are crucial for many physiological and pathological processes
Through extensive and time-consuming efforts of large-scale preparation of highly homogeneous kindlins and subsequent crystallization trials, we were able to crystallize human full-length kindlin-3 that was expressed as a single monomeric species in bacterial Escherichia coli BL21 system
We determined the first structure of human full-length kindlin-3, using the selenine (Se)–single-wavelength anomalous diffraction (SAD) phasing method in combination with Phenix program (Paul Adams, Randy Read, Jane & Dave Richardson, Tom Terwilliger; https://www.phenix-online.org/)

Summary

Introduction

Integrin-mediated cell–cell and cell–extracellular matrix interactions are crucial for many physiological and pathological processes. The 4.1 protein, ezrin, radixin and moesin (FERM)-domain–containing kindlin family of proteins has emerged as key players involved in integrin activation. The importance of kindlins is underscored by their link to Kindler syndrome (a disorder within the spectrum of hereditary epidermolytic blistering diseases), leukocyte adhesion deficiency (LAD) III (a hereditary primary immunodeficiency with platelet dysfunction disease), and cancer [2,3]. The sequence homology between kindlin-1 and kindlin-2 is 62%, and those between kindlin-1 and kindlin-3, as well as kindlin-2 and kindlin-3 are 54% and 52%, respectively (44% for all 3). In addition to sequence similarity, all kindlins share atypical FERM domain organization comprising the F1, F2, and F3 subdomains (Fig 1A and S1 Fig).

Methods

Results

Conclusion