Unidirectional peristaltic movement in multisite drug binding pockets of AcrB from molecular dynamics simulations

Tripartite complex AcrB-ToIC, the major efflux system in Escherichia coli, is the principal multidrug transporter in Gram-negative bacteria, which is important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel ToIC and the membrane fusion protein AcrA. Recently, the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin were reported. Here we performed 20 ns molecular dynamics (MD) simulations of the AcrB-rifampicin-minocycline complex in a lipid bilayer and explicit water. We found that the bound drugs, rifampicin and erythromycin, made a unidirectional peristaltic movement towards the extrusion funnel of ToIC, which was facilitated by the water efflux in the channel of AcrB. With a shift of the Phe-617 loop, rifampicin in the access monomer moved towards the entrance of the distal binding pocket. Minocycline in the binding monomer moved from the distal binding pocket towards the gate of the central funnel. The channel between the entrance and the gate made a concerted opening during the MD simulations, which was helpful for the peristaltic movement. Our results showed that the mutations of Gly616Pro and Gly619Pro prevented the movement of the Phe-617 loop, which indicated the critical role of the flexibility of the Phe-617 loop. In addition, three putative proton translocation channels were proposed based on our results. Our study provided dynamical information and important residues for the peristaltic movement in AcrB, which were critical for substrate uptake and extrusion function.     

Substrate-dependent transport mechanism in AcrB of multidrug resistant bacteria

The multidrug resistance (MDR) system effectively expels antibiotics out of bacteria causing serious issues during bacterial infection. In addition to drug, indole, a common metabolic waste of bacteria, is expelled by MDR system of gram-negative bacteria for their survival. Experimental results suggest that AcrB, one of the key components of MDR system, undergoes large scale conformation changes during the pumping due to proton-motive process. However, due to extremely short time scale, it is difficult to observe (experimentally) those changes in the AcrB, which might facilitate the pumping process. Molecular simulations can shed light to understand the conformational changes for transport of indole in AcrB. Examination of conformational changes using all-atom simulation is, however, impractical. Here, we develop a hybrid coarse-grained force field to study the conformational changes of AcrB in presence of indole in the porter domain of monomer II. Using the coarse-grained force field, we investigated the conformational changes of AcrB for a number of model systems considering the effect of protonation in aspartic acid (Asp) residues Asp407 and Asp408 in the transmembrane domain of monomer II. Our results show that in the presence of indole, protonation of Asp408 or Asp407 residue causes conformational changes from binding state to extrusion state in monomer II, while remaining two monomers (I and III) approach access state in AcrB protein. We also observed that all three AcrB monomers prefer to go back to access state in the absence of indole. Steered molecular dynamics simulations were performed to demonstrate the feasibility of indole transport mechanism for protonated systems. Identification of indole transport pathway through AcrB can be very helpful in understanding the drug efflux mechanism used by the MDR bacteria.     

Water molecules and hydrogen-bonded networks in bacteriorhodopsin--molecular dynamics simulations of the ground state and the M-intermediate

Protein crystallography provides the structure of a protein, averaged over all elementary cells during data collection time. Thus, it has only a limited access to diffusive processes. This article demonstrates how molecular dynamics simulations can elucidate structure-function relationships in bacteriorhodopsin (bR) involving water molecules. The spatial distribution of water molecules and their corresponding hydrogen-bonded networks inside bR in its ground state (G) and late M intermediate conformations were investigated by molecular dynamics simulations. The simulations reveal a much higher average number of internal water molecules per monomer (28 in the G and 36 in the M) than observed in crystal structures (18 and 22, respectively). We found nine water molecules trapped and 19 diffusive inside the G-monomer, and 13 trapped and 23 diffusive inside the M-monomer. The exchange of a set of diffusive internal water molecules follows an exponential decay with a 1/e time in the order of 340 ps for the G state and 460 ps for the M state. The average residence time of a diffusive water molecule inside the protein is approximately 95 ps for the G state and 110 ps for the M state. We have used the Grotthuss model to describe the possible proton transport through the hydrogen-bonded networks inside the protein, which is built up in the picosecond-to-nanosecond time domains. Comparing the water distribution and hydrogen-bonded networks of the two different states, we suggest possible pathways for proton hopping and water movement inside bR.     

Water wires in atomistic models of the Hv1 proton channel

The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.     

The mechanism of proton translocation in respiratory complex I from molecular dynamics

Abstract

Respiratory complex I, the biggest enzyme of respiratory chain, plays a key role in energy production by the mitochondrial respiratory chain and has been implicated in many human neurodegenerative diseases. Recently, the crystal structure of respiratory complex I is reported. We perform 50?ns molecular dynamics simulations on the membrane domain of respiratory complex I under two hypothetical states (oxidized state and reduced state). We find that the density of water molecules in the trans-membrane domain under reduced state is bigger than that under oxidized state. The connecting elements (helix HL and β-hairpins-helix element) fluctuate stronger under reduced state than that under oxidized state, causing more internal water molecules and facilitating the proton conduction. The conformational changes of helix HL and the crucial charged residue Glu in TM5 play key roles in the mechanism of proton translocation. Our results illustrate the dynamic behavior and the potential mechanism of respiratory complex I, which provides the structural basis for drug design of respiratory complex I.     

How environment supports a state: molecular dynamics simulations of two states in bacteriorhodopsin suggest lipid and water compensation

The light-driven proton pump bacteriorhodopsin (bR) is a transmembrane protein that uses large conformational changes for proton transfer from the cytoplasmic to the extracellular regions. Crystal structures, due to their solvent conditions, do not resolve the effect of lipid molecules on these protein conformational changes. To begin to understand the molecular details behind such large conformational changes, we simulated two conformations of wild-type bacteriorhodopsin, one of the dark-adapted state and the second of an intermediate (M(O)) state, each within an explicit dimyristoyl-phosphatidylcholine (DMPC) lipid bilayer. The simulations included all-hydrogen and all-atom representations of protein, lipid, and water and were performed for 20 ns. We investigate the equilibrium properties and the dynamic motions of the two conformations in the lipid setting. We note that the conformational state of the M(O) intermediate bR remains markedly different from the dark-adapted bR state in that the M(O) intermediate shows rearrangement of the cytoplasmic portions of helices C, F, and G, and nearby loops. This difference in the states remained throughout the simulations, and the results are stable on the molecular dynamics timescale and provide an illustration of the changes in both lipid and water that help to stabilize a particular state. Our analysis focuses on how the environment adjusts to these two states and on how the dynamics of the helices, loops, and water molecules can be related to the pump mechanism of bacteriorhodopsin. For example, water generally behaves in the same manner on the extracellular sides of both simulations but is decreased in the cytoplasmic region of the M(O) intermediate. We suspect that the different water behavior is closely related to the fluctuations of microcavities volume in the protein interior, which is strongly coupled to the collective motion of the protein. Our simulation result suggests that experimental observation can be useful to verify a decreased number of waters in the cytoplasmic regions of the late-intermediate stages by measuring the rate of water exchange with the interior of the protein.     

Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.     

Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined. Analysis of the amino acid sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to approximately 15 A as opposed to the previously reported value of approximately 30 A for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.     

Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli.

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.     

Molecular dynamics studies on structure and dynamics of phospholamban monomer and pentamer in membranes

Phospholamban (PLB) is an integral membrane protein of 52 residues that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle cells through reversible phosphorylation of Ser16. To explore its possible conformations and dynamics in a monomeric state, we have performed comparative molecular dynamics simulations of unphosphorylated and phosphorylated PLB (pPLB) with various orientations in POPC membranes. The simulations indicate that dynamics of the cytoplasmic domain is highly dependent on its interactions with membranes, that is, large conformational changes in the absence of membrane interactions, but very restricted dynamics in their presence. pPLB shows more structural flexibility in its cytoplasmic domain, which is consistent with experimental observations. We have also performed a simulation of a PLB pentameric structure (the so‐called bellflower model), recently determined in micelles, to investigate its behaviors in a POPC membrane. The cytoplasmic domain in each monomer shows uncorrelated dynamics and undergoes large conformational changes toward the membrane surface during the simulation, which supports the so‐called pinwheel model of the PLB pentamer structure. The hydrophobic nature of the pentameric pore excludes water molecules in the pore region, which illustrates that the pore appears to be an energetic barrier for ion and water translocation. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ammonium recruitment and ammonia transport by E. coli ammonia channel AmtB

To investigate substrate recruitment and transport across the Escherichia coli Ammonia transporter B (AmtB) protein, we performed molecular dynamics simulations of the AmtB trimer. We have identified residues important in recruitment of ammonium and intraluminal binding sites selective of ammonium, which provide a means of cation selectivity. Our results indicate that A162 guides translocation of an extraluminal ammonium into the pore lumen. We propose a mechanism for transporting the intraluminally recruited proton back to periplasm. Our mechanism conforms to net transport of ammonia and can explain why ammonia conduction is lost upon mutation of the conserved residue D160. We unify previous suggestions of D160 having either a structural or an ammonium binding function. Finally, our simulations show that the channel lumen is hydrated from the cytoplasmic side via the formation of single file water, while the F107/F215 stack at the inner-most part of the periplasmic vestibule constitutes a hydrophobic filter preventing AmtB from conducting water.     

Understanding structure and function in the light-driven proton pump bacteriorhodopsin

The atomic structure of bacteriorhodopsin and the outlines of its proton transport mechanism are now available. Photoisomerization of the retinal in the chromophore creates a steric and electrostatic conflict at the retinal binding site. The free energy gain sets off a sequence of reactions in which directed proton transfers take place between the protonated retinal Schiff base, Asp-85, and Asp-96. These internal steps, and other proton transfers at and near the two aqueous interfaces, add up to the translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Bound water plays a crucial role in proton conduction in both extracellular and cytoplasmic regions, but the means by which the protons move from site to site differ. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. The directionality of the translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96.     

Multidrug efflux transporter, AcrB--the pumping mechanism

Resistance nodulation cell division (RND) transporters are one of the main causes of the bacterial multidrug resistance. They pump a wide range of antibiotics out of the cell by proton motive force. AcrB is the major RND transporter in Escherichia coli. Recently, the crystal structures of AcrB have been determined by different space groups. All these structures are consistent with asymmetric trimer. Each monomer has different conformation corresponding to one of the three functional states of the transport cycle. Transporting hydrophobic drug was bound in the periplasmic domain on one of the three monomers. The transport pathway with alternating access mechanism is located at the hydrophilic domain protruded into the periplasmic space while this mechanism of other transporter families like ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporter is located in the membrane-embedded region. For the RND, protonation might also take place asymmetrically at the functionally important charged residues in the transmembrane (TM) region. The structures indicate that drugs are transported by a three-step functional rotation in which substrates undergo ordered binding change.     

New insights into the structural and functional involvement of the gate loop in AcrB export activity

AcrB is a major multidrug exporter in Escherichia coli and other Gram-negative bacteria. Its gate loop, located between the proximal and the distal pockets, have been reported to play important role in the export of many antibiotics. This loop location, rigidity and interactions with substrates have led recent reports to suggest that AcrB export mechanism operates in a sequential manner. First the substrate binds the proximal pocket in the access monomer, then it moves to bind the distal pocket in the binding monomer and subsequently it is extruded in the extrusion monomer. Recently, we have demonstrated that the gate loop is not required for the binding of Erythromycin but the integrity of this loop is important for an efficient export of this substrate. However, here we show that the antibiotic susceptibilities of the same AcrB gate loop mutants for Doxorubicin were unaffected, suggesting that this loop is not required for its export, and we demonstrate that this substrate may use principally the tunnel-1, located between transmembranes 8 and 9, more often than previously reported. To further explain our findings, here we address the gate loop mutations effects on AcrB solution energetics (fold, stability, molecular dynamics) and on the in vivo efflux of Erythromycin and Doxorubicin. Finally, we discuss the efflux and the discrepancy between the structural and the functional experiments for Erythromycin in these gate loop mutants.     

Multidrug-exporting secondary transporters

The major cause of intrinsic drug resistance in Gram-negative bacteria is a resistance nodulation division type multidrug exporter, which couples with an outer membrane channel and a membrane fusion protein and exports drugs out of the cell, bypassing the periplasm; this process is driven by proton motive force. A recent crystal structure determination of a major resistance nodulation division type multidrug exporter, AcrB in Escherichia coli, greatly advances our understanding of the multidrug export mechanism. The most striking feature of the AcrB trimer is the presence of three vestibules open to the periplasm at the boundary between the periplasmic headpiece and the transmembrane region. Substrates can gain access to the central cavity from the periplasmic surface of the cytoplasmic membrane and are then actively transported through the extramembrane pore into the outer membrane channel TolC, via the funnel at the top of the AcrB headpiece.     

Cytoplasmic shuttling of protons in anabaena sensory rhodopsin: implications for signaling mechanism

It was found recently that Anabaena sensory rhodopsin (ASR), which possibly serves as a photoreceptor for chromatic adaptation, interacts with a soluble cytoplasmic transducer. The X-ray structure of the transducer-free protein revealed an extensive hydrogen-bonded network of amino acid residues and water molecules in the cytoplasmic half of ASR, in high contrast to its haloarchaeal counterparts. Using time-resolved spectroscopy of the wild-type and mutant ASR in the visible and infrared ranges, we tried to determine whether this hydrogen-bonded network is used to translocate protons and whether those proton transfers are important for interaction with the transducer. We found that the retinal Schiff base deprotonation, which occurs in the M intermediate of the photocycle of all-trans-ASR, results in protonation of Asp217 on the cytoplasmic side of the protein. The deprotonation of the Schiff base induces a conformational change of ASR observed through the perturbation of associated lipids. We suggest that the cytoplasmic shuttling of protons in the photocycle of all-trans-ASR and the ensuing conformational changes might activate the transducer. Consequently, the M intermediate may be the signaling state of ASR.     

Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway

We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.     

Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin

The L intermediate in the proton-motive photocycle of bacteriorhodopsin is the starting state for the first proton transfer, from the Schiff base to Asp85, in the formation of the M intermediate. Previous FTIR studies of L have identified unique vibration bands caused by the perturbation of several polar amino acid side chains and several internal water molecules located on the cytoplasmic side of the retinylidene chromophore. In the present FTIR study we describe spectral features of the L intermediate in D(2)O in the frequency region which includes the N-D stretching vibrations of the backbone amides. We show that a broad band in the 2220-2080 cm(-1) region appears in L. By use of appropriate (15)N labeling and mutants, the lower frequency side of this band in L is assigned to the amides of Lys216 and Gly220. These amides are coupled to each other, and interact with Thr46 and Val49 in helix B and Asp96 in helix C via weakly H-bonding water molecules that exhibit O-D stretching vibrations at 2621 and 2605 cm(-1). These water molecules are part of a hydrogen-bonded network characteristic of L which includes other water molecules located closer to the chromophore that exhibit an O-D stretching vibration at 2589 cm(-1). This structure, extending from the Schiff base to the internal proton donor Asp96, stabilizes L and affects the L-to-M transition.     

Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.