Structural basis of cooperativity in kinesin revealed by 3D reconstruction of a two-head-bound state on microtubules.

Daifei Liu,Xueqi Liu,Zhiguo Shang,Charles V Sindelar

doi:10.7554/elife.24490

Abstract

The detailed basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. Utilizing novel electron cryomicroscopy methods, we solved structures of microtubule-attached, dimeric kinesin bound to an ATP analog. We find that under these conditions, the kinesin dimer can attach to the microtubule with either one or two motor domains, and we present sub-nanometer resolution reconstructions of both states. The former structure reveals a novel kinesin conformation that revises the current understanding of how ATP binding is coupled to forward stepping of the motor. The latter structure indicates how tension between the two motor domains keeps their cycles out of phase in order to stimulate directional motility. The methods presented here pave the way for future structural studies of a variety of challenging macromolecules that bind to microtubules and other filaments.

Highlights

Kinesins are molecular motor proteins that use the energy of ATP hydrolysis to move unidirectionally along microtubules
We developed a new method, FINDKIN, which is capable of locating bound kinesin motors on microtubules and has allowed us to determine the first structure of a kinesin dimer whose two motor domains bind at sequential sites along a single protofilament
In order to assess the efficacy of FINDKIN, we first applied it to synthetic microtubules, where the exact locations of bound kinesin motors are known and individual parameters, such as image noise level, occupancy and defocus can be controlled (Figure 2—figure supplement 1)

Summary

Introduction

Kinesins are molecular motor proteins that use the energy of ATP hydrolysis to move unidirectionally along microtubules. Dozens of kinesin proteins have been found in humans, functioning in a wide variety of biological processes, from cellular cargo transport to mitosis (Rath and Kozielski, 2012). The kinesin-1 motor domain, which houses both ATPase and microtubule-binding activities, is located at its N-terminal end. Kinesin-1 forms a homodimer via the formation of a coiled coil by the stalk domains in the middle of the polypeptide chains. Kinesin-1 homodimer takes eight nanometer steps through head-over-head movements, advancing its two motor domains alternatively along a single protofilament (Gennerich and Vale, 2009)

Methods

Results

Conclusion