All-Atom Structural Investigation of Kinesin–Microtubule Complex Constrained by High-Quality Cryo-Electron-Microscopy Maps

Abstract

In this study, we have performed a comprehensive structural investigation of three major biochemical states of a kinesin complexed with microtubule under the constraint of high-quality cryo-electron-microscopy (EM) maps. In addition to the ADP and ATP state which were captured by X-ray crystallography, we have also modeled the nucleotide-free or APO state for which no crystal structure is available. We have combined flexible fitting of EM maps with regular molecular dynamics simulations, hydrogen-bond analysis, and free energy calculation. Our APO-state models feature a subdomain rotation involving loop L2 and α6 helix of kinesin, and local structural changes in active site similar to a related motor protein, myosin. We have identified a list of hydrogen bonds involving key residues in the active site and the binding interface between kinesin and microtubule. Some of these hydrogen bonds may play an important role in coupling microtubule binding to ATPase activities in kinesin. We have validated our models by calculating the binding free energy between kinesin and microtubule, which quantitatively accounts for the observation of strong binding in the APO and ATP state and weak binding in the ADP state. This study will offer promising targets for future mutational and functional studies to investigate the mechanism of kinesin motors.

Decrypting the Structural, Dynamic, and Energetic Basis of a Monomeric Kinesin Interacting with a Tubulin Dimer in Three ATPase States by All-Atom Molecular Dynamics Simulation

“…We found fewer HBs in ADP state, and more HBs in APO and ATP state than ref 56. As a result, the new HB counts are consistent with weak (strong) MT binding in ADP state (APO and ATP state), while the old HB counts in ref 56 are not.…”

“…(5) In the analysis of kinesin−tubulin binding free energy, the new ADP state exhibited weaker binding than in ref 56. This difference is probably due to the use of long unconstrained MD simulation here vs short constrained MD simulation in ref 56.…”

“…Such difference in MT-binding affinity is essential to drive structural changes that release ADP, facilitate ATP binding and hydrolysis, and generate force. In our previous studies, 55,56 we established and applied an MDbased binding free energy calculation protocol (see Methods) to the kinesin−tubulin complex which yielded lower (more negative) binding free energy (denoted ΔG) for APO and ATP Table S6). This is because the unrestrained kinesin is more mobile during the new longer MD simulation of ADP state, so it can move further away from tubulin dimer, resulting in weaker binding.…”

All‐atom molecular dynamics simulations reveal how kinesin transits from one‐head‐bound to two‐heads‐bound state

“…Using all‐atom MD simulations, the dynamics of the NL docking, which is involved in the forward stepping of the motor, was studied . Detailed structural and dynamic pictures of a kinesin head in nucleotide‐free, ADP and ATP states bound to MT tubulin were studied . The interactions between MT tubulin and a kinesin head in different nucleotide states were analyzed and the binding energies were calculated .…”

Molecular dynamics simulation of tropomyosin bound to actins/myosin in the closed and open states

“…We used a continuum solvent model to estimate the protein‐protein binding free energy, which was previously established for motor proteins and Tpm . Following our previous studies, we used the MD‐generated conformations to estimate the Tpm ‐ actins/myosin binding free energy Δ G for the central Tpm fragment and its constituent periods P3‐P6.…”