Abstract
(+)-Morelloflavone (MF) is an antitumor biflavonoid that is found in the Garcinia species. Recently, we reported MF as a novel inhibitor of ATPase and microtubules-gliding activities of the kinesin spindle protein (Eg5) in vitro. Herein, we provide dynamical insights into the inhibitory mechanisms of MF against Eg5, which involves binding of the inhibitor to the loop5/α2/α3 allosteric pocket. Molecular dynamics simulations were carried out for 100 ns on eight complexes: Eg5-Adenosine diphosphate (Eg5-ADP), Eg5-ADP-S-trityl-l-cysteine (Eg5-ADP-STLC), Eg5-ADP-ispinesib, Eg5-ADP-MF, Eg5-Adenosine triphosphate (Eg5-ATP), Eg5-ATP-STLC, Eg5-ATP-ispinesib, and Eg5-ATP-MF complexes. Structural and energetic analyses were done using Umbrella sampling, Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) method, GROMACS analysis toolkit, and virtual molecular dynamics (VMD) utilities. The results were compared with those of the known Eg5 inhibitors; ispinesib, and STLC. Our data strongly support a stable Eg5-MF complex, with significantly low binding energy and reduced flexibility of Eg5 in some regions, including loop5 and switch I. Furthermore, the loop5 Trp127 was trapped in a downward position to keep the allosteric pocket of Eg5 in the so-called “closed conformation”, comparable to observations for STLC. Altered structural conformations were also visible within various regions of Eg5, including switch I, switch II, α2/α3 helices, and the tubulin-binding region, indicating that MF might induce modifications in the Eg5 structure to compromise its ATP/ADP binding and conversion process as well as its interaction with microtubules. The described mechanisms are crucial for understanding Eg5 inhibition by MF.
Highlights
Kinesins are among the most explored protein families in protein–ligand interaction studies, probably owing to their important roles in essential cellular processes, such as cargo transport, neuronal development, and cell division
Eg5-Adenosine diphosphate (Eg5-adenosine diphosphate (ADP))-MF, Eg5-ADP-STLC, and Eg5-ADP-ispinesib complexes are mainly stabilized by electrostatic, van der Waals, and polar solvation energies
As MF was previously found to inhibit Eg5 activities, we investigated the dynamics of the Eg5-MF interaction at the molecular level using atomistic simulation to explain the inhibitory mechanism of the biflavonoid
Summary
Kinesins are among the most explored protein families in protein–ligand interaction studies, probably owing to their important roles in essential cellular processes, such as cargo transport, neuronal development, and cell division. As early as 20 ns into the simulation, the allosteric pocket of Eg5, in the absence of inhibitors (Eg5-ADP), began to open (as deduced from the eventual distance between residues Trp127 and Glu215) reaching 2.31 ± 0.2 Å, and failed to close until the end of the simulation (Figure 4A). For Eg5-ATP complex, the loop5/α2/α3 pocket opened as early as 3.5 ns during the simulation, with an attempt to close at approximately 20 ns This attempted closure was promptly reversed and the distance increased to 3.44 Å. This observation on the loop5/α2/α3 pocket of Eg5 was further substantiated by evaluating the distance between Tyr211 and Trp127 The distance between these residues decreased significantly in Eg5-ADP-MF and Eg5-ADP-STLC structures compared to Eg5 structures in the absence of inhibitors (Figure 4C), suggesting the trapping of loop in the downward position. This behavior marks the likely similar mode of action of these molecules, which is compatible with their similar binding configuration [29]
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