Cryo-EM structure of the β-1,3-glucan synthase FKS1-Rho1 complex

Abstract

Abstract The β-1,3 glucan synthase (GS) is essential for the biosynthesis of β-1,3 glucan, a well-conserved structural component of fungal cell wall. The GS holoenzyme is a multi-enzyme complex consisting of the glycosyltransferase FKS and the essential regulatory factor Rho1, a small GTPase. However, the precise mechanism by which Rho1 activates FKS1 activity in a GTP-dependent manner remains elusive. Here, we present two cryo-electron microscopy (cryo-EM) structures of FKS1 alone (resting state) and FKS1-Rho1 complex (activating state), respectively. Structural analysis reveals that FKS1 adopts a cellulase-like conformation, wherein two segments of the cytoplasmic domain tightly bound together to form a functional structural unit. Remarkably, we unveil that the interaction between Rho1 and FKS1 is enhanced in the presence of a nonhydrolyzable guanosine triphosphate analog (GTP-γ-S). Rho1 is positioned within a pocket between the cytoplasmic domain of FKS1 and the transmembrane helix spanning TM7-15, engaging with the highly conserved glycosyltransferase domain of FKS1 (GT domain). Comparative analysis between the unbound (resting state) and Rho1-bound structures of FKS1 reveals the extensive conformational changes within FKS1, specifically in the GT domain and TM7-15. These alterations suggest that Rho1's GTP/GDP cycling acts as a molecular pump, inducing a dynamic transition between the resting and activating states of FKS1. Notably, the activation of Rho1 triggering FKS1 conformation changes, an evolutionary conserved "finger helix" within the FKS1-Rho1 complex adopts an up-and-down movement, ultimately pushing the growing glucan chain into FKS1’s transmembrane channel, thereby facilitating β-1,3 glucan elongation. Collectively, our results provide a vivid ratchet and pawl model to describe the mechanism of fungal β-1,3-glucan biosynthesis.