| Intramembrane protease is crucial for many biological and pathological processes as a special life regulator,it evolves independently and reacts within the lipid membrane,which contributes to the process of intercellular signaling delivery and membrane protein quality control.Accelerating the discoveries of biological roles of these proteases can make a progress in some disease treatments,including Alzheimer's disease,Parkinson's disease,cancer,malaria infection,hepatitis C virus maturation,tuberculosis virulence,and diabetes.Meanwhile,it was reported that the rhomboid proteases can regulate functional processes,such as mitochondrial dynamics and regulation of apoptotic stimuli.Recently,the crystal structure of rhomboid-substrate?complex?has been resolved,which provides chance to explore unknown molecular mechanism.The structure of rhomboid protease is composed of six helixes and four loops mainly,and embedded in the lipid bilayer.Two catalytic residues are located in Tm4 and Tm6,respectively.Notably,the catalytic residue Ser201 is sunk 10?into the plane of the membrane in a hydrophilic cavity.Previous studies have shown that the substrate is further stabilized in the active site by hydrogen bonds of its backbone with the residues Ser248 and Ala250 of the Loop5,and residues Gly198 and Trp196 of the Loop3;Side chain and main chain atoms of the substrate can engage in van der Waals interactions with residues of the Loop3?P1 alanine and Gly199,P3 alanine and Phe197?and the Loop5?P2 threonine and Met249?.However,due to characteristic of the protease structure,the pathway of substrate entry active site as key component of molecular mechanism has not yet been described in detail.In this study,the molecular dynamics simulations were performed for the apoenzyme and complex in the POPE lipid bilayers to obtain possible pathways for substrate entry.Based on the binding site of substrate in protein,two possible pathways were selected.The pathway 1 is located in bottom of protein and between Tm5 and Tm2,and the direction of the pathway 1 is determined by the vector from the C?of Ala5 to C?of Gly7 of substrate.Meanwhile,the pathway 2 is in top of protein between Loop5 and Loop3,and the direction of the pathway 2 is determined by the vector from the C?of Thr3 to C?of Ile1.The substrate expulsion along the two directions is the reverse process of substrate binding along two directions.Therefore,they should have the same energy changes and conformational states.As reverse process of substrate entry,the steered molecular dynamics simulations were carried out to pull the substrate from the active site,which can simulate process of substrate entry.Based on the result of steered molecular dynamics,umbrella sampling simulations were performed.Finally,the potential of mean force?PMF?profiles reveal energy changes of substrate unbinding were 34.71 and 34.44 kcal·mol-1 along the pathway 1 and pathway 2,respectively.Therefore,the more feasible pathway was not selected certainly from the results of numerical values of energy changes.Further,the previous research revealed that the membrane environment slows proteolytic rate,in part,by restraining protease gate opening.The direction of pathway 1 was between Tm2 and Tm5,which was located in middle of lipid bilayer,while the direction of pathway 2 was upward,and there were fewer lipid barriers at the exit of pathway 2.Therefore,the pathway 2 still might be more feasible than pathway 1.When the substrate accessed active site along pathway 2,Tm2,Loop3 and Loop5,Tm5 had conformational change in order to accommodate substrate in the binding groove.Besides,His150 as important residue could make substrate conformation change and adapt channel of protein,and could assist substrate binding.These important conclusions can promote the development of the catalytic mechanism of rhomboid protease and the design and synthesis of it's selective inhibitor. |
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