The mechanism of proton exclusion in the aquaporin-1 water channel

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.     

Substrate versus inhibitor dynamics of P‐glycoprotein

By far the most studied multidrug resistance protein is P‐glycoprotein. Despite recent structural data, key questions about its function remain. P‐glycoprotein (P‐gp) is flexible and undergoes large conformational changes as part of its function and in this respect, details not only of the export cycle, but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P‐gp. In agreement with previous reports, we found that P‐gp undergoes large conformational changes which tended to result in the nucleotide‐binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to previous cross‐linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds bound with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart, thus preventing ATP‐hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P‐gp. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Molecular dynamics simulation of dark-adapted rhodopsin in an explicit membrane bilayer: coupling between local retinal and larger scale conformational change

The light-driven photocycle of rhodopsin begins the photoreceptor cascade that underlies visual response. In a sequence of events, the retinal covalently attached to the rhodopsin protein undergoes a conformational change that communicates local changes to a global conformational change throughout the whole protein. In turn, the large-scale protein change then activates G-proteins and signal amplification throughout the cell. The nature of this change, involving a coupling between a local process and larger changes throughout the protein, may be important for many membrane proteins. In addition, functional work has shown that this coupling occurs with different efficiency in different lipid settings. To begin to understand the nature of the efficiency of this coupling in different lipid settings, we present a molecular dynamics study of rhodopsin in an explicit dioleoyl-phosphatidylcholine bilayer. Our system was simulated for 40 ns and provides insights into the very early events of the visual cascade, before the full transition and activation have occurred. In particular, we see an event near 10 ns that begins with a change in hydrogen bonding near the retinal and that leads through a series of coupled changes to a shift in helical tilt. This type of event, though rare on the molecular dynamics time-scale, could be an important clue to the types of coupling that occur between local and large-scale conformational change in many membrane proteins.     

Structure refinement of membrane proteins via molecular dynamics simulations

A refinement protocol based on physics‐based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge‐based or implicit membrane‐based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid‐facing residues. Scoring with knowledge‐based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane‐based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Accurate and efficient description of protein vibrational dynamics: comparing molecular dynamics and Gaussian models

Current all-atom potential based molecular dynamics (MD) allows the identification of a protein's functional motions on a wide-range of timescales, up to few tens of nanoseconds. However, functional, large-scale motions of proteins may occur on a timescale currently not accessible by all-atom potential based MD. To avoid the massive computational effort required by this approach, several simplified schemes have been introduced. One of the most satisfactory is the Gaussian network approach based on the energy expansion in terms of the deviation of the protein backbone from its native configuration. Here, we consider an extension of this model that captures in a more realistic way the distribution of native interactions due to the introduction of effective side-chain centroids. Since their location is entirely determined by the protein backbone, the model is amenable to the same exact and computationally efficient treatment as previous simpler models. The ability of the model to describe the correlated motion of protein residues in thermodynamic equilibrium is established through a series of successful comparisons with an extensive (14 ns) MD simulation based on the AMBER potential of HIV-1 protease in complex with a peptide substrate. Thus, the model presented here emerges as a powerful tool to provide preliminary, fast yet accurate characterizations of protein near-native motion.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.     

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4×20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a ‘closed’ and an ‘open’ pore-like state of the carrier protein.     

Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core

The folding of most integral membrane proteins follows a two‐step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four‐helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six‐helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD‐simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process.     

One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

Membrane protein dynamics versus environment: simulations of OmpA in a micelle and in a bilayer

The bacterial outer membrane protein OmpA is one of the few membrane proteins whose structure has been solved both by X-ray crystallography and by NMR. Crystals were obtained in the presence of detergent, and the NMR structure is of the protein in a detergent micelle. We have used 10 ns duration molecular dynamics simulations to compare the behaviour of OmpA in a detergent micelle and in a phospholipid bilayer. The dynamic fluctuations of the protein structure seem to be ca 1.5 times greater in the micelle environment than in the lipid bilayer. There are subtle differences between the nature of OmpA-detergent and OmpA-lipid interactions. As a consequence of the enhanced flexibility of the OmpA protein in the micellar environment, side-chain torsion angle changes are such as to lead to formation of a continuous pore through the centre of the OmpA molecule. This may explain the experimentally observed channel formation by OmpA.     

Mutagenesis of the putative nucleotide-binding domains of the multidrug resistance associated protein (MRP). Analysis of the effect of these mutations on MRP mediated drug resistance and transport

Studies were conducted to examine the functional role of the nucleotide-binding domains of MRP in drug resistance and drug transport in isolated membrane vesicles. In vivo studies were conducted by preparing stable transfectants of HeLa cells with wild-type MRP cDNA or MRP cDNAs which had been mutated at certain nucleotide binding domains (NBD). Stable transfectants producing equivalent amounts of the MRP encoded protein P190 were used in this study. The results demonstrated that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to chemotherapeutic agents. Certain single-site mutations in lysines in these same motifs also reduce IC₅₀ values. It has also been observed that mutation of the MRP NBDs results in an increase in drug accumulation and a reduction in drug efflux. Additional studies have been carried out in which recombinant baculovirus containing either wild-type MRP or MRP containing mutated NBDs was prepared and used to infect SF21 insect cells. Using this system we have analyzed the effects of these mutations on in vitro transport of leukotriene C₄ (LTC₄) 17 β-estradiol 17 (β-D-glucuronide)(E₂17βG) and daunomycin in membrane vesicles prepared from baculovirus infected cells. The results demonstrate that deletions and site-specific mutations in MRP NBDs greatly reduce the ATP dependent transport of all three substrates. The results of these studies conducted both in vivo and in vitro demonstrate that the NBDs of MRP function in a cooperative manner and are critical for the transport activity of the MRP encoded protein P190. These studies also identify specific lysines in NBD1 and NBD2 which are important for optimal MRP activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Crystal structure of the Campylobacter jejuni CmeC outer membrane channel

As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance‐nodulation‐cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export.     

Water dynamics simulation as a tool for probing proton transfer pathways in a heptahelical membrane protein

The proton transfer pathway in a heptahelical membrane protein, the light-driven proton pump bacteriorhodopsin (BR), is probed by a combined approach of structural analysis of recent X-ray models and molecular dynamics (MD) simulations that provide the diffusion pathways of internal and external water molecules. Analyzing the hydrogen-bond contact frequencies of the water molecules with protein groups, the complete proton pathway through the protein is probed. Beside the well-known proton binding sites in the protein interior-the protonated Schiff base, Asp85 and Asp96, and the H(5)O(2) (+) complex stabilized by Glu204 and Glu194-the proton release and uptake pathways to the protein surfaces are described in great detail. Further residues were identified, by mutation of which the proposed pathways can be verified. In addition the diffusion pathway of water 502 from Lys216 to Asp96 is shown to cover the positions of the intruding waters 503 and 504 in the N-intermediate. The transiently established water chain in the N-state provides a proton pathway from Asp96 to the Schiff base in the M- to N-transition in a Grotthus-like mechanism, as concluded earlier from time-resolved Fourier transform infrared experiments [le Coutre et al., Proc Nat Acad Sci USA 1995;92:4962-4966].     

Diffusional interaction behavior of NSAIDs in lipid bilayer membrane using molecular dynamics (MD) simulation: Aspirin and Ibuprofen

In this research, for the first time, molecular dynamics (MD) method was used to simulate aspirin and ibuprofen at various concentrations and in neutral and charged states. Effects of the concentration (dosage), charge state, and existence of an integral protein in the membrane on the diffusion rate of drug molecules into lipid bilayer membrane were investigated on 11 systems, for which the parameters indicating diffusion rate and those affecting the rate were evaluated. Considering the diffusion rate, a suitable score was assigned to each system, based on which, analysis of variance (ANOVA) was performed. By calculating the effect size of the indicative parameters and total scores, an optimum system with the highest diffusion rate was determined. Consequently, diffusion rate controlling parameters were obtained: the drug–water hydrogen bond in protein-free systems and protein–drug hydrogen bond in the systems containing protein.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Molecular dynamics simulations of microstructure and mixing dynamics of cryoprotective solvents in water and in the presence of a lipid membrane

Molecular dynamics (MD) simulation is used to investigate the solubility behavior of cryoprotective (CP) solvents, such as DMSO, ethylene glycol (EG) and glycerol (GL), in pure water and in the presence of a lipid membrane. The MD study is focused on an equilibration timescale required for mixing large CP aggregates with aqueous and aqueous/lipid environments. The MD analysis demonstrates that DMSO mixes rapidly with water, so that all solute molecules are uniformly distributed in the equilibrium aqueous solution. Our investigation of the microstructure of binary EG/water and GL/water systems reveals that, despite the miscibility of both CP solvents with water, they are not ideally mixed in aqueous solutions at the molecular level. The MD simulations show that the mixing dynamics of the large CP cluster and surrounding water is found to be strongly dependent on nature of hydrophilic and hydrophobic interactions acting between cryoprotectant molecules. In particular, a spatial hydrogen-bond network formed between CP molecules plays an important role in the mixing dynamics between CP agents and water. A further analysis on the mixing behavior of the CP solvents with pure water and with aqueous solutions at a lipid membrane interface shows that, due to strong binding of the CP molecules to membrane surface, the equilibration process in the lipid environment becomes very slow, at least of the order of microseconds. The MD results are discussed in the context of the better understanding on the composition of the aqueous mixtures of the EG and GL solvents. Knowledge of the microstructure and the dynamics of these systems helps to develop better cryopreservation protocols and to propose more optimal cooling/warming regimes for cellular cryosolutions.     

Molecular dynamics characterisations of the Trp-cage folding mechanisms: in the absence and presence of water solvents

Protein folding is an important and yet challenging topic in current molecular biology. In this work, the folding dynamics and mechanisms of the Trp-cage mini-protein were studied with molecular dynamics simulations, in the absence and presence of water solvents. The important intermediates during the Trp-cage folding were determined by gradually decreasing the simulation temperature. The folding transition temperature was identified to be approximately 400 K, and the folding pathway was decomposed into six steps: U ? I ₁ ? I ₂ ? I ₃ ? I ₄ ? F ₁ ? F ₂, where U, I and F represent the unfolded, intermediate and folded states, respectively. The finding that the two helical subunits are successively formed is consistent with the experimental observations, and the Asp9/Arg16 salt bridge forms at the final stage and does not play a significant role during folding kinetics. The presence of water solvents induces hydrophobic collapse as the whole cage comparatively closes. Within aqueous solutions, the Trp-cage folding begins to contract into the meta-stable state, and by traversing the transition state it arrives at the native-like structure, which resembles the experimental structure closely.     

Molecular dynamics simulation: A new way to understand the functionality of the endothelial glycocalyx

Endothelial glycocalyx (EG) is a carbohydrate-rich layer which lines the lumen side of blood vessel walls. The EG layer is directly exposed to blood flow. The unique physiological location and its strongly coupled interaction with blood flow allow the EG layer to modulate microvascular mass transport and to sense and transmit mechanical signals from the passing blood. Molecular dynamics (MD) simulation is a computational method which focuses on atomic/molecular behavior at the microscale. The last two decades have witnessed a substantial increase in number and a broadening in scope regarding applications of MD in a wide spectrum of areas, including EG-related research. In this mini-review, MD works which solve EG-related problems and provide new insights into the functionality of EG are considered. Challenges of the MD method in EG research are articulated, and the future of MD in solving EG-related problems is also evaluated.