Getting in Sync with Dimeric Eg5: INITIATION AND REGULATION OF THE PROCESSIVE RUN

Troy C Krzysiak,Michael Grabe,Susan P Gilbert

doi:10.1074/jbc.m708354200

Abstract

Eg5/KSP is the kinesin-related motor protein that generates the major plus-end directed force for mitotic spindle assembly and dynamics. Recent work using a dimeric form of Eg5 has found it to be a processive motor; however, its mechanochemical cycle is different from that of conventional Kinesin-1. Dimeric Eg5 appears to undergo a conformational change shortly after collision with the microtubule that primes the motor for its characteristically short processive runs. To better understand this conformational change as well as head-head communication during processive stepping, equilibrium and transient kinetic approaches have been used. By contrast to the mechanism of Kinesin-1, microtubule association triggers ADP release from both motor domains of Eg5. One motor domain releases ADP rapidly, whereas ADP release from the other occurs after a slow conformational change at approximately 1 s(-1). Therefore, dimeric Eg5 begins its processive run with both motor domains associated with the microtubule and in the nucleotide-free state. During processive stepping however, ATP binding and potentially ATP hydrolysis signals rearward head advancement 16 nm forward to the next microtubule-binding site. This alternating cycle of processive stepping is proposed to terminate after a few steps because the head-head communication does not sufficiently control the timing to prevent both motor domains from entering the ADP-bound state simultaneously.

Highlights

Eg5 is a homotetrameric Bim C/kinesin-5 family member that plays a vital role in the mitotic spindle and has attracted substantial interest as a potential target for chemotherapeutic agents in cancer treatment [1,2,3,4,5,6,7,8,9]
This study has revealed that dimeric Eg5 begins a processive run from a nucleotide-free state with both motor domains associated with the MT
Our initial kinetic analysis of Eg5-513 suggested that steadystate ATP turnover by dimeric Eg5 is governed by a slow structural transition [35, 43]

Summary

EXPERIMENTAL PROCEDURES

Standard Conditions—The experiments were performed at 25 °C in ATPase buffer: 20 mM Hepes, pH 7.2, with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM dithiothreitol. The excluded protein was subjected to a second round of gel filtration in ATPase buffer to remove the EDTA and to adjust the solution back to standard conditions with 5 mM magnesium acetate Both protocols to obtain nucleotide-free Eg5-513NF yielded kinetics that were indistinguishable experimentally. To determine the kinetics of mantADP release from the high affinity site, the experiment was repeated using the Eg51⁄7mantADP complex obtained after gel filtration. After rapid mixing in the stopped flow, the fluorescence enhancement of mantATP binding to the active site was monitored (Fig. 3).

RESULTS

(Supplemental Material and Table

DISCUSSION