High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration

Abstract

In Escherichia coli, the binding of the chemotaxis signaling protein CheY to the flagellar motor protein FliM induces reversal of the motor rotation. Using molecular dynamics (MD) simulations and parallel cascade selection MD (PaCS-MD), we show that high pressure increases the water density in the first hydration shell of CheY and considerably induces water penetration into the CheY-FliM interface. PaCS-MD enabled us to observe pressure-induced dissociation of the CheY-FliM complex at atomic resolution. Pressure dependence of binding free energy indicates that the increase of pressure from 0.1 to 100 MPa significantly weakens the binding. Using high-pressure microscopy, we observed that high hydrostatic pressure fixes the motor rotation to the counter-clockwise direction. In conclusion, the application of pressure enhances hydration of the proteins and weakens the binding of CheY to FliM, preventing reversal of the flagellar motor.Pressure significantly affects protein structure, dynamics, and functions 1-4 . Very high pressure (above 500 MPa) induces denaturation of many soluble proteins 5-8 , and therefore the mechanism of pressure denaturation has been relatively well investigated, both theoretically and experimentally 4,9,10 . On the other hand, pressures below 100 MPa, a range that covers the typical biosphere on earth, including the deep sea, does not induce large changes in overall secondary and tertiary structure of most protein molecules 1,3,11,12 , but was found to affect behaviors of biological systems at macroscopic levels [13][14][15] . Pressures of ~ 100 MPa are known to interfere with interactions between biomolecules such as proteins and ligands 1,4,14,16,17 , indicating that the mechanism whereby high pressure perturbs such interactions should be elucidated at the molecular level.Molecular dynamics (MD) simulation has been applied to obtain atomic details of structures and functions of biomolecules, including surrounding water and other molecules 18-20 . MD simulations under high pressures have observed enhanced hydration of proteins 21-23 and penetration of water molecules inside proteins with accompanying conformational changes 24,25 . However, it remains difficult to simulate pressure effects on protein-protein interactions, given that the timescale for observing the pressure effects is often longer than the length of typical MD simulations 4,26 . Recently, a new distributed computing method, parallel cascade selection molecular dynamics (PaCS-MD), has been developed to observe events whose timescales are longer than the standard MD length 27,28 . PaCS-MD can simulate dissociation process of protein complexes, whose time scales range from μs to s, without using artificial forces within a short MD simulation time 29 . Additionally, by integrating the distributed MD trajectories using a Markov state model (MSM) 30 , various quantities characterizing molecular interactions can be calculated, such as binding free energy, association/dissociation rate constants, and residence times 29,31 .In the pr...

Binding Free Energy of Protein/Ligand Complexes Calculated using Dissociation Parallel Cascade Selection Molecular Dynamics and Markov State Model

“…The dissociation of protein/ligand complexes simulated using PaCS-MD [56] according to the procedure was described earlier [28]. Ten parallel MDs (replicas) were used for the trypsin/benzamine and FKBP/FK506 complexes and 30 replicas were employed for A2A/T4E.…”

“…PaCS-MD comprises cycles of multiple parallel short (typically 0.1 ns) MD simulations combined with initial structure selection. The repetition of parallel MD simulations from selected promising structures with regenerated initial atom velocities drastically enhances the probability of observing the dissociation of protein/ligand complexes by selecting snapshots with longer protein-ligand distances [26][27][28].…”

“…A dynamic description of simulated unbinding processes can be obtained by constructing an MSM from many short PaCS-MD trajectories. By selecting snapshots with longer intermolecular distances, our group previously used dissociation PaCS-MD (dPaCS-MD) to generate dissociation pathways of tri-N-acetyl-dglucosamine from hen egg white lysozyme [26], of the transactivation domain of p53 protein (p53-TAD) from murine double-minute clone 2 protein (MDM2) protein [30], and of an N-terminal fragment of the bacterial flagellar rotor protein FliM from the signaling protein CheY [28]. We built an MSM model using dPaCS-MD trajectories to investigate the dissociation mechanisms by describing states based on ligand/protein geometry [29,31].…”

Session 1SHA—control of biological functions with hydrostatic pressure stimulation

“…Ikuro Kawagishi (Hosei University) showed that pressure affects bacterial chemotaxis. He demonstrated that high hydrostatic pressure can induce counter-clockwise rotation of the flagellar motor of Escherichia coli (Hata et al 2020a). Katsumi Imada (Osaka University) reported the molecular mechanisms of pressure-dependent fluorescence change of a yellow fluorescent protein mutated by the insertion of three glycine residues at β7 (YFP-3G) (Watanabe et al 2013).…”

“…Katsumi Imada (Osaka University) reported the molecular mechanisms of pressure-dependent fluorescence change of a yellow fluorescent protein mutated by the insertion of three glycine residues at β7 (YFP-3G) (Watanabe et al 2013). Hiroaki Hata (Tokyo Institute of Technology) indicated that pressure enhanced hydration around proteins can inhibit the protein-protein interactions that play a key role in bacterial chemotaxis (Hata et al 2020a). In addition, Oleg Dobrokhotov (Nagoya University) showed that artificial induction of actomyosin-generated tension inhibits proliferation and promotes differentiation of keratinocyte carcinoma cells.…”