A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

Abstract

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

Electrostatics of folded and unfolded bovine β-lactoglobulin

“…For evaluating this possibility, the flexibility of the 3BLG C terminus was investigated at an atomic level through a LowModeMD approach, which has already been successfully used for studying the P-loop conformations of Rho-kinase (Gohda and Hakoshima 2008) and the c-jun N-terminal kinase JNK3 (Kolossváry and Keserü 2001). This approach allowed us to sample some 3BLG C terminus conformations with a non-buried His161, associated with pK a similar to the model value (Table 2), which suggests that His161 pK a value seems to be very sensitive to the local conformation.…”

LowModeMD—Implicit Low-Mode Velocity Filtering Applied to Conformational Search of Macrocycles and Protein Loops

“…The right-hand side of Figure 2 depicts the superimposed complexes of 2F2U with complex A in green and complex B in orange. In a recent study, Gohda et al 38 attempted, but failed, to interconvert the conformations using 2 ns of molecular dynamics; they concluded that a much longer simulation would be required to observe a loop conformation change. We applied 1000 iterations of Low-ModeMD to this task, attempting to convert conformation A into B vice versa.…”

Synthesis and Biological Evaluation of Novel (thio)semicarbazone-Based Benzimidazoles as Antiviral Agents against Human Respiratory Viruses

“…Finally, the reliability of the selected docking poses was assessed using a short~1 ps run of molecular dynamics (MD) at constant temperature, followed by an all-atom energy minimization (LowModeMD implemented in MOE software). This represents a conformational search method that uses implicit vibrational analysis to focus a MD trajectory along the low-mode vibrations [41][42][43]. This has the effect of searching for minima along the valleys and troughs on the potential energy surface, thereby performing an exhaustive conformational analysis of the ligand-receptor binding site complex, as we previously discussed about other case studies [44][45][46].…”