Structural Basis of Microtubule Polymerases in Accelerating Tubulin Polymerization via Multiple Tog Domains

Abstract

XMAP215/Dis1 proteins are microtubule polymerases that accelerate αβ-tubulins assembly at microtubule plus ends via multiple conserved Tumor Overexpressed Gene (TOG) domains; however, their mechanisms remains poorly understood. Here, we describe structural basis for microtubule polymerase using biochemistry and x-ray crystallography by studying multi-TOG domain-αβ-tubulin complexes in two states along the polymerization pathway. We show TOG1 and TOG2 domains bind αβ-tubulins but release them with different rates using biochemical methods. Upon inhibiting αβ-tubulin polymerization, we determine x-ray structures revealing that two subunits of TOG1-TOG2 domains form dimeric head-to-tail square assemblies bound to αβ-tubulins, stabilized by novel higher-order TOG domain interfaces. In this intermediate the four αβ-tubulins are positioned in a polarized organization, 90°-rotated from polymerization contacts. Upon controlled release of αβ-tubulin polymerization, TOG2 dissociate from TOG1 domains, rotating and polymerizing αβ-tubulins to form a head-to-tail two-subunit polymer. A new paradigm emerges from our studies to explain microtubule polymerase catalysis suggesting two TOG1-TOG2 domain subunits cooperatively organize αβ-tubulins in a polarized organization prior to polymerization, and dissociation of TOG domains at microtubule plus ends leads tubulin polymerization due to the organization coupled with an αβ-tubulin TOG domain release rate gradient, with the most rapid being at the outermost end of a newly formed polymer.