Insight into microtubule nucleation from tubulin-capping proteins.

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or ß-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit.

Microtubules are cytoskeletal filaments of eukaryotic cells made of αß-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation.

Insight into microtubule disassembly by kinesin-13s from the structure of Kif2C bound to tubulin.

Kinesin-13s are critical microtubule regulators which induce microtubule disassembly in an ATP dependent manner. To clarify their mechanism, we report here the crystal structure of a functional construct of the kinesin-13 Kif2C/MCAK in an ATP-like state and bound to the αß-tubulin heterodimer, a complex mimicking the species that dissociates from microtubule ends during catalytic disassembly. Our results picture how Kif2C stabilizes a curved tubulin conformation. The Kif2C α4-L12-α5 region undergoes a remarkable 25° rotation upon tubulin binding to target the αß-tubulin hinge. This movement leads the ß5a-ß5b motif to interact with the distal end of ß-tubulin, whereas the neck and the KVD motif, two specific elements of kinesin-13s, target the α-tubulin distal end. Taken together with the study of Kif2C mutants, our data suggest that stabilization of a curved tubulin is an important contribution to the Kif2C mechanism.Kinesin-13s are microtubule depolymerizing enzymes. Here the authors present the crystal structure of a DARPin fused construct comprising the short neck region and motor domain of kinesin-13 in complex with an αß-tubulin heterodimer, which shows that kinesin-13 functions by stabilizing a curved tubulin conformation.

The structural switch of nucleotide-free kinesin.

Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.