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

Weiyi Wang,Qiyang Jiang,Manuela Argentini,David Cornu,Benoît Gigant,Marcel Knossow,Chunguang Wang

doi:10.1074/jbc.m111.317859

Abstract

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Highlights

Kinesin-13 proteins, such as Kif2C, share a conserved motor domain with motile kinesins, but they depolymerize microtubules
Comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension
We studied the binding of nucleotides to this protein and, to relate it to the depolymerase function, we measured the affinities of Kif2C-(sNϩM) in its three main nucleotide states for MTs and tubulin

Summary

Background

Kinesin-13 proteins, such as Kif2C, share a conserved motor domain with motile kinesins, but they depolymerize microtubules. Comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension. We studied the binding of nucleotides to this protein and, to relate it to the depolymerase function, we measured the affinities of Kif2C-(sNϩM) in its three main nucleotide states (nucleotide-free, ADP-bound, and ATP-bound) for MTs and tubulin. Because in addition the affinities for tubulin of the Kif2C motor domain in its three main nucleotide states are much more similar than those of Kif2C-(sNϩM), our results suggest that the increased specificity for curved tubulin of Kif2C-(sNϩM) in the ATP-bound state enhances the depolymerase activity and that this enhancement is an important role of the short Neck extension. Its function is to detach the kinesin from curved, longitudinally associated tubulin heterodimers, which thereby dissociate from each other and from MTs

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION