The lattice as allosteric effector: Structural studies of αβ- and γ-tubulin clarify the role of GTP in microtubule assembly

Abstract

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, ␣␤-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in ␣␤-tubulin. The allosteric model posits that unpolymerized ␣␤-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of ␣␤-tubulin, the 2.3 Å crystal structure of ␥-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous ␥-tubulin:GTP␥S structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of ␣␤-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive ␣␤-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. ␣␤-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior. dynamic instability ͉ microtubules M icrotubules are hollow cylindrical polymers of ␣␤-tubulin that are critical for intracellular trafficking and formation of the mitotic spindle required for chromosome segregation during cell division. Although vitally important, the molecular mechanisms underlying dynamic microtubule (MT) behavior are poorly understood. Microtubule assembly requires GTP-bound ␣␤-tubulin, and GTP hydrolysis by ␤-tubulin is required to generate dynamic MTs (reviewed in ref. 1). Structural studies have demonstrated two extreme conformations of ␣␤-tubulin: a ''straight'' conformation observed in the MT lattice (2), and a "curved" conformation observed in a structure of ␣␤-tubulin complexed with colchicine and a stathmin-like domain (3) (Fig. 1). There are two opposing models for the relationship between GTP and conformational change in ␣␤-tubulin. The allosteric model (recently reviewed in ref. 4) postulates that GTP binding to ␣␤-tubulin triggers long-range conformational changes yielding a substantially straighter conformation that is prestructured in solution for lateral interactions (5). The lattice model (6) postulates that in solution, ␣␤-tubulin adopts the MTincompatible, curved conformation independent of nuc...

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

“…For example, if the protofilaments are curved at the microtubule end, a tensile force will tend to straighten the protofilament and this may facilitate the incorporation of a "bent" dimer into the straight lattice (46,48). However, we estimate that a 1 pN force will have only a very modest effect on the curvature of a protofilament, ( 1 degree per tubulin dimer (using the flexural rigidity of a single protofilament used in ref.…”

“…A third possibility is that a tensile force induces a structural change at the microtubule end that facilitates polymerization (45)(46)(47)(48)(49). For example, if the protofilaments are curved at the microtubule end, a tensile force will tend to straighten the protofilament and this may facilitate the incorporation of a "bent" dimer into the straight lattice (46,48).…”

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

“…To be competent for assembly, tubulin has to be in the GTP state (i.e., with GTP bound to the ␤-subunit). There is increasing evidence that unassembled GTP-tubulin is curved, one of the most compelling arguments being that it binds allocolchicine and several other colchicine-domain ligands with an affinity very similar to that of GDP-tubulin (21,22). The question then arises of how GTP favors tubulin assembly in microtubules.…”

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

“…Initial modeling based on the x-ray structure of a kinesin-13 construct has suggested that it would be better adapted to binding to curved tubulin, a property to which the class-specific L2 loop and its KVD motif might contribute (20). It is important to challenge this at the biochemical level by comparing the affinities of kinesins-13 for microtubular tubulin (which is straight) and for the soluble tubulin heterodimer (which is curved) (21) and to determine the dependence of any preference on the nucleotide state of the kinesin (ATP-bound, ADP-bound or nucleotide-free). The second aspect concerns one of the most fundamental aspects of the depolymerization mechanism, the role of ATP hydrolysis.…”