Nucleotide-dependence of G-actin conformation from multiple molecular dynamics simulations and observation of a putatively polymerisation-competent superclosed state

The assembly of monomeric G-actin into filamentous F-actin is nucleotide dependent: ATP-G-actin is favored for filament growth at the “barbed end” of F-actin, whereas ADP-G-actin tends to dissociate from the “pointed end.” Structural differences between ATP- and ADP-G-actin are examined here using multiple molecular dynamics simulations. The “open” and “closed” conformational states of G-actin in aqueous solution are characterized, with either ATP or ADP in the nucleotide binding pocket. With both ATP and ADP bound, the open state closes in the absence of actin-bound profilin. The position of the nucleotide in the protein is found to be correlated with the degree of opening of the active site cleft. Further, the simulations reveal the existence of a structurally well-defined, compact, “superclosed” state of ATP-G-actin, as yet unseen crystallographically and absent in the ADP-G-actin simulations. The superclosed state resembles structurally the actin monomer in filament models derived from fiber diffraction and is putatively the polymerization competent conformation of ATP-G-actin

A seesaw model for intermolecular gating in the kinesin motor protein

Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement

Molecular dynamics study of the energetic, mechanistic, and structural implications of a closed phosphate tube in ncd.

The switch 1 region of myosin forms a lid over the nucleotide phosphates as part of a structure known as the phosphate-tube. The homologous region in kinesin-family motors is more open, not interacting with the nucleotide. We used molecular dynamics (MD) simulations to examine a possible displacement of switch 1 of the microtubule motor, ncd, from the open conformation to the closed conformation seen in myosin. MD simulations were done of both the open and the closed conformations, with either MgADP or MgATP at the active site. All MD structures were stable at 300 K for 500 ps, implying that the open and closed conformers all represented local minima on a global free energy surface. Free energy calculations indicated that the open structure was energetically favored with MgADP at the active site, suggesting why only the open structure has been captured in crystallographic work. With MgATP, the closed and open structures had roughly equal energies. Simulated annealing MD showed the transformation from the closed phosphate-tube ncd structure to an open configuration. The MD simulations also showed that the coordination of switch 1 to the nucleotide dramatically affected the position of both the bound nucleotide and switch 2 and that a closed phosphate-tube may be necessary for catalysis

A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATIP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P-i) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site

A comparative study of motor-protein motions by using a simple elastic-network model

In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996) Phys. Rev. Lett. 77, 1905–1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the “nucleotide-binding-induced power-stroke” scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities