CONFORMATION & RECOGNITION IN MICROTUBLE DYNAMICS

Ut Southwestern Medical Center, Dallas TX

Investigators

Linked publications & trials

Abstract

Microtubules (MTs) are essential dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. It is increasingly appreciated that the polymerizing ??-tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to promote elongation, shrinking, or catastrophe. However, integrating these structural and biochemical findings into a mechanistic understanding of MT dynamics and regulation remains a central challenge. In the prior project period, we pioneered a powerful approach based on structure-inspired site- directed ??-tubulin mutants. Using these methods, we advanced the understanding of the regulatory MT polymerase Stu2p by discovering that two simple concepts ? selective binding to a MT-incompatible conformation of ??-tubulin and tethering-based concentration of reactants ? could explain the catalytic action of the polymerase. We also advanced the understanding of MT dynamics, discovering by studying buried mutations in ?-tubulin that a tunable allosteric response to GDP in the lattice dictates the frequency of MT catastrophe and the rate of post-catastrophe shrinking. Our laboratory is now uniquely positioned to answer fundamental questions about MT dynamics and regulation. In Aim 1 we will use a combination of protein engineering and in vitro reconstitution, including single molecule experiments, to answer most of the remaining questions about the mechanism of MT polymerases: the molecular origin of processivity, what determines the degree of polymerase saturation on the MT end, and how maximal polymerase activity depends on the number and type of TOG domains. This will result in a state-of-the-art understanding of a MT regulatory protein that integrates structure and biochemistry with bulk and single-molecule kinetic results In Aim 2 we capitalize on our finding that ?:E255A, a surface mutation at the site of GTPase activity, causes a `straightening defect'. We will use mutagenesis, measurements of MT dynamics, negative stain and cryo electron microscopy, and other approaches to discover the mechanism of assembly-dependent ??-tubilin straightening, identify allosteric coupling within the heterodimer, and show how they contribute to MT dynamics and the structure of ??-tubulin assemblies. In Aim 3 we will use a stable of new reagents to determine ??-tubulin structures without binding partners or bound to a TOG domain. This work will answer longstanding questions about conformation(s) of unpolymerized ??-tubulin, will reveal whether allosteric mutations change it, and it will clarify what ??-tubulin conformations can be recognized by TOG domains. Our approach is distinctive and promises to deliver previously unobtainable insight into fundamental mechanisms of MT dynamics and regulation.