Interaction of the Eg5 Loop 5 with the Nucleotide Binding Site

Abstract

Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the P-loop at the nucleotide site of all kinesin super-family motors. In kinesin-1 and kinesin-3 motors, L5 is 6-8 amino acids in length. Kinesin-5 motors such as Eg5 have longer L5 loops, ∼17 a.a. in length. X-ray structures show that L5 is the binding site for small molecules that inhibit microtubule-stimulated ADP release by Eg5. However, crystallography has failed to identify the function of L5 because all Eg5 structures, both with and without bound inhibitors, show similar conformations for L5. It is bent away from the nucleotide site with an unusual loop W127 residue interacting with hydrophobic surface patches and the inhibitor. The proximity of the Eg5 L5 to the nucleotide site suggests it could interact with a bound nucleotide, modulating function. Larson (this meeting) presents EPR spectroscopy data supporting this conclusion. We have used molecular modeling and molecular dynamics (MD) simulations to investigate the potential interaction of L5 and the nucleotide. The L5 domain of the Eg5•ADP x-ray structure was manually deformed via phi-psi backbone rotations. L5 was sufficiently lengthy that W127 could be located in proximity to the adenine ring of ADP. The modified structure was solvated in a box of explicit waters and energy minimized. After 1000 ps of MD simulation, a stable structure was obtained. The structure shows L5 interacting with the adenine ring of ADP via W127 in a pocket formed by the hydrophobic portions of the side chains of E129, D130, and by P27. The structure shows significant impingement on the ribose hydroxyls, consistent with the experimental results of Larson. Thus the simulations provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.