Molecular Dynamics Simulation Study Of Transmembrane Signal:GPCR-Arrestin Complex And Mechanosensitive Ion Channel OSCA

Signal transduction through cell membranes is a fundamental procedure in living organisms.Membrane proteins play a fundamental role in transmembrane signal transduction,which can sense external stimuli and transmit the signal to intracellular downstream effectors.Therefore,membrane proteins can regulate multiple biological processes such as cognition,immune response,blood pressure regulation,nociception and vascular development.Because of its indispensable function,membrane proteins attract much attention.Here,we investigated the signal transduction mechanisms of two membrane proteins,belonging to G-protein coupled receptors(GPCR)and mechanosensitive ion channels respectively,which represent the two major families of membrane proteins.GPCRs serve as the most important drug targets at present.Over 27%of the available drugs target GPCR to cure diseases.Arrestin forms a small family of proteins which can bind to GPCR,block G protein interactions and redirect signaling to G-protein-independent pathways.The detailed mechanism of how arrestin interacts with GPCR remains elusive.Based on the complex structure of arrestin binding with rhodopsin(a typical class A GPCR),we conducted molecular dynamics(MD)simulations to investigate the stability and orientation of the two proteins upon the complex formation.Our results showed that the complex formation has little impact on the orientation of rhodopsin while influences the arrestin's orientation to a large extent.Further analyses show that there are multiple non-bonded interactions in the complex interface,which can stabilize the structure of the rhodopsin-arrestin complex.These results help us to better understand how arrestin interacts with GPCR on membranes,and thereby shed light on arrestin-mediated signal transduction through GPCRs.Different external stimuli may make GPCR recruit different intracellular effectors through arrestin.What's the mechanism of this selective signaling?One hypothesis is that different stimuli can lead to distinct phosphorylation patterns in the GPCR C-terminus loop(C-loop),and arrestin will adopt specific conformations in accord with the phosphorylation pattern of the GPCR C-loop when forming a complex with it and then recruit various downstream signaling molecules.To validate this hypothesis,we performed MD simulations to investigate the conformational changes of arrestin induced by different rhodopsin C-loop phosphorylation pattens.Our results showed that arrestin experienced significant conformational changes when binding to rhodopsin.The C-domain of the activated arrestin showed an obvious twist in comparison with the apo-state conformation,while the twist angle is similar in all of the simulations with different C-loop phosphorylation states,indicating that the global conformational change of arrestin upon different C-loop phosphorylation states is not distinguishable in our current simulations.To further explore the correlation between the phosphorylation states of the GPCR C-loop and the arrestin conformation,we analyzed the pair-wise force distribution between the phosphorylated residues of C-loop and the neighboring residues of the arrestin N-domain,which showed that these forces are highly depended on the phosphorylation patterns.Comparing the force distribution in different phosphorylation states,we found that two residues,T336rhodopsin and S338rhodopsin,which are located at the middle position of the rhodopsin C-loop,play a fundamental role in the GPCR C-loop and the arrestin interaction.When T336rhodopsin and S338rhodopsin are phosphorylated,the interaction between pT336rhodopsin-R172arrestin and pS338rhodopsin-K301 arrestin may serve as anchors that make the C-loop tightly attach to the N-domain of arrestin.When the T340rhodopsin,T342rhodopsin and S343rhodopsin are phosphorylated,the neighboring residues of arrestin N-domain can sense the perturbation and pass it to adjacent residues.Eventually,the phosphorylation pattern can probably influence the downstream protein binding site by allosteric effect.This mechanism can help to explain why distinct phosphorylation patterns can drive arrestin into unique conformations and result in selective binding of downstream signaling molecules.Therefore,our results shed new lights on the understanding of the phosphorylation pattern dependent signaling through the GPCR-arrestin pathway.Mechanosensitive ion channels play a vital role in diverse mechanotransduction processes.AtOSCA1(Arabidopsis thaliana reduced hyperosmolality induced[Ca2+]increase 1,hereinafter called "OSCA"),a typical mechanosensitive ion channel in plants,can respond to the force from membranes induced by the changes of the osmotic pressure and serve as an osmosensor of plants.Recently,a Cryo-EM OSCA structure was resolved,which showed an interesting dimeric architecture.Because of the complexity of the structure,one cannot determine the ion permeation pore and the gating mechanism from the sole structure.To solve these questions,we performed multi-scale MD simulations based on the OSCA structure.The results showed that the lipid molecules can assemble around the OSCA to form a bilayer and some lipid molecules spontaneously occupied the cavity between the two subunits of OSCA to block the water and ions permeation.Intriguingly,we found transient and continuous water distribution inside each subunit,which is considered as the precondition of ions permeation to occur.Therefore,our results indicated that the permeation pore is probably located inside each OSCA subunit rather than the cavity between the two subunits.We also investigated the activation mechanism of OSCA.As demonstrated by previous experiments,OSCA should be responsive to mechanical forces from lipids.We applied a 50-mN/m surface tension to the lipid bilayer surrounding the OSCA structure in our MD simulations,and OSCA responded to this mechanical stimulus and the pore radius extended significantly in the MD trajectories.Accordingly,water molecules fulfilled the pore and formed a continuous and steady water distribution pathway.Analyzing the pore dilation process in our simulation trajectories,we spotted two regions that may restrain the motion of the transmembrane helixes M4 and M6 upon the pore opening.One is the hydrophobic network at the upper region of the pore and the other is a salt bridge(K436M4-D524M6)in the lower region.Interrupting these two interactions may facilitate the OSCA activation.Based on our MD results,we proposed a plausible activation mechanism of OSCA:The most outward transmembrane helix M0 senses the forces from lipids and moves away from the center of the pore,which provides more space for M6 to fluctuate and move away from M4.The motion of M6 causes collapse of the hydrophobic network in the upper pore and water molecules flow into the pore.As a consequence,the pore is further dilated and the OSCA channel is gradually activated.