Jingwei Weng

The Conformational Transition Pathways of ATP-Binding Cassette Transporter BtuCD Revealed by Targeted Molecular Dynamics Simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B12 into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the “alternating access” mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O→I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I→O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process.

Interdomain Flexibility and pH-Induced Conformational Changes of AcrA Revealed by Molecular Dynamics Simulations

The membrane fusion protein (MFP) AcrA is proposed to link the inner membrane transporter AcrB and outer membrane protein TolC, forming the tripartite AcrAB-TolC efflux pump, and was shown to be functionally indispensible. Structural and EPR studies showed that AcrA has high conformational flexibility and exhibited pH-induced conformational change. In this study, we built the complete structure of AcrA through homology modeling and performed atomistic simulations of AcrA at different pH values. It was shown that the conformational flexibility of AcrA originates from the motions of α-hairpin and MP domains. The conformational dynamics of AcrA is sensitive to specific point mutations and pH values. In agreement with the EPR experiments, the interdomain motions were restrained upon lowering pH from 7.0 to 5.0 in the simulations. It was found that the protonation/deprotonation of His285 underlies the pH-regulated conformational dynamics of AcrA by disturbing the local hydrogen bond interactions, suggesting that the changes of pH in the periplasm accompanying the drug efflux could act as a signal to trigger the action of AcrA, which undergoes reversible conformational rearrangement.

Detailed regulatory mechanism of the interaction between ZO-1 PDZ2 and connexin43 revealed by MD simulations.

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation.

Insights into the Inhibitory Mechanism of D13-9001 to the Multidrug Transporter AcrB through Molecular Dynamics Simulations.

The resistance-nodulation-cell division transporter AcrB is responsible for energy transduction and substrate recognition in the tripartite AcrAB-TolC efflux system in Escherichia coli. Despite a broad substrate specificity, only a few compounds have been cocrystallized with AcrB inside the distal binding pocket (DBP), including doxorubicin (DOX) and D13-9001. D13-9001 is a promising efflux pump inhibitor that potentiates the efficacy of a wide variety of antibiotics. To understand its inhibition effect under the framework of functional rotating mechanism, we performed targeted and steered molecular dynamics simulations to compare the binding and extrusion processes of this inhibitor and the substrate DOX in AcrB. The results demonstrate that, with respect to DOX, the interaction of D13-9001 with the hydrophobic trap results in delayed disassociation from the DBP. Notably, the detachment of D13-9001 is tightly correlated with the side-chain reorientation of Phe628 and large-scale displacement of Tyr327. Furthermore, the inhibitor induces much more significant conformational changes at the exit gate than DOX does, thereby causing higher energy cost for extrusion and contributing to the inhibitory effect in addition to the tight binding at DBP.

Molecular Dynamics Simulation of Membrane Proteins

Membrane proteins play crucial roles in a range of biological processes. High resolution structures provide insights into the functional mechanisms of membrane proteins, but detailed biophysical characterization of membrane proteins is difficult. Complementary to experimental techniques, molecular dynamics simulations is a powerful tool in providing more complete description of the dynamics and energetics of membrane proteins with high spatial-temporal resolution. In this chapter, we provide a survey of the current methods and technique issues for setting up and running simulations of membrane proteins. The recent progress in applying simulations to understanding various biophysical properties of membrane proteins is outlined.

Elastic network model‐based normal mode analysis reveals the conformational couplings in the tripartite AcrAB‐TolC multidrug efflux complex

The AcrAB‐TolC drug efflux system, energized by proton movement down the transmembrane electrochemical gradient, is responsible for the resistance of the organism to a wide range of drugs. Experimental data suggest functional roles of each part of the assembly, but the detailed working mechanism of this machinery remains elusive. We used elastic network‐based normal mode analysis (NMA) to explore the conformational dynamics of the AcrAB‐TolC complex. The intrinsic flexibilities of the pore domain in AcrB monomer conform to the previously proposed three‐step functionally rotating mechanism for asymmetric AcrB trimer. Conformational couplings across monomers in the AcrB trimer were observed, and the coupling between the transmembrane domain and the other parts of AcrB are strengthened through trimeric assembly. In the tripartite AcrAB‐TolC assembly obtained through molecular docking, concerted motions were observed not only at the direct contact interfaces between various components but also between distant parts of the whole complex. The presence of AcrA was shown to significantly strengthen the motional couplings between AcrB and TolC. Overall, NMA revealed an allosteric network in the AcAB‐TolC efflux system, which provides hints to our understanding of its detailed working mechanism. Proteins 2011;

Stepwise substrate translocation mechanism revealed by free energy calculations of doxorubicin in the multidrug transporter AcrB.

AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.

Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB

The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states [access (A), binding (B) and extrusion (E)] support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE to BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB.

Lipid Regulated Conformational Dynamics of the Longin SNARE Protein Ykt6 Revealed by Molecular Dynamics Simulations

The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the farnesylated state of Ykt6. In this study, we performed in silico farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the farnesylated Ykt6 model structure is stable during the 2 μs simulation and the farnesyl group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study.

Mechanism of Ser88 phosphorylation-induced dimer dissociation in dynein light chain LC8.

Dynein light chain LC8 is a highly conserved, dimeric protein involved in a variety of essential cellular events. Phosphorylation at Ser88 was found to promote mammalian cell survival and regulate the dimer to monomer transition at physiological pH. Combining molecular dynamics (MD) simulation and free energy calculation methods, we explored the atomistic mechanism of the phosphorylation-induced dimer dissociation. The MD simulation revealed that phosphorylation/phosphomimetic mutation at Ser88 opens an entrance into the dimer interface for water molecules, which disturb the hydrogen bond network around His55 and is expected to raise the pK(a) value and protonation ratio of His55 as well. The free energy calculations showed that the S88E mutation destabilized the dimer by 6.6 kcal/mol, in good agreement with the experimental value of 8.1 kcal/mol. The calculated destabilization upon phosphorylation is 50.8 kcal/mol, showing that phosphorylation definitely prevents dimer formation under physiological conditions. Further analysis of the calculated free energy changes demonstrated that the electrostatic contribution dominates the impact of phosphorylation on dimer dissociation.