Coiled-coil domain kinking controls laminin-332 cleavage by elastase

Abstract

Laminins are trimeric glycoproteins and essential components of basement membranes, where they provide structural support and mediate cell-matrix adhesion. Their -, {beta}-, and {gamma}-chains combine into a flexible trimeric coiled-coil domain forming the laminin long arm, which links the N-terminal short arms and the C-terminal globular domains. However, ultrastructural insight into the laminin coiled-coil domain remains limited, and the function of coiled-coil flexibility remains unclear. Using high-speed atomic force microscopy (HS-AFM) imaging, we previously investigated the ultrastructure of laminin-332 and observed dynamic kinking around a putative molecular hinge in the center of the coiled-coil domain. In this study, we integrate HS-AFM time-lapse imaging with AlphaFold structure prediction and normal-mode flexible fitting (NMFF) to generate dynamic models of the laminin-332 hinge during coiled-coil domain kinking, providing atomic-scale insight into the underlying molecular rearrangements. Furthermore, we use HS-AFM to visualize, in real time, the digestion of individual laminin-332 molecules by pancreatic elastase and show that coiled-coil kinking directs elastase cleavage to the hinge site, thereby ensuring reliable generation of the elastase 8 (E8) fragment containing the integrin binding site. In contrast, laminin molecules with extended coiled-coil conformations are cleaved at arbitrary sites, resulting in complete coiled-coil removal and degradation. Our results thus identify a novel conformational control mechanism directing proteolytic processing of laminin-332 through defined coiled-coil kinking. Besides expanding the understanding of dynamic laminin coiled-coil function, the gained ultrastructural insight into trimeric coiled-coil bending may aid the rational design of flexible coiled-coil modules for future protein engineering applications.