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Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi-drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter species) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of ‘molecular guns’ known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation-cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research, in silico studies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein–lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria.

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β2-adrenergic receptor (β2 AR). The 3.2 A X-ray crystal structure of human β2 AR active-state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of L-DOPA and Droxidopa into rigid and flexible β2 AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, re-docking neutral and protonated versions of adrenaline to rigid and flexible β2 AR models results in binding poses that are more energetically stable and distinct from the X-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β2 AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and Droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the MD simulations of β2AR-ligand complexes performed here. SIGNIFICANCE The β2-adrenergic receptor (β2 AR), which belongs to the vast family of Guanine nucleotide-binding protein coupled receptors (GPCRs), is a transmembrane protein that is activated by the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Using molecular docking methods, we discovered that the exogenous ligands L-DOPA and Droxidopa have the same hydrogen bond binding sites as endogenous agonists such as adrenaline, noradrenaline, and dopamine. Furthermore, all of the catecholamines studied had different hydrophobic binding sites in the receptor. Our findings might help researchers to better understand how existing and new drugs chemically similar to Droxidopa, which is used to treat Parkinson's disease, interact with β2 AR.

The secondary transporters of the resistance-nodulation-cell division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria like Pseudomonas aeruginosa. Among these RND transporters, MexB, MexF, and MexY, with partly overlapping specificities, have been implicated in pathogenicity. Only the structure of the former has been resolved experimentally, which together with the lack of data about the functional dynamics of the full set of transporters, limited a systematic investigation of the molecular determinants defining their peculiar and shared features. In a previous work (Ramaswamy et al., Front. Microbiol., 2018, 9, 1144), we compared at an atomistic level the two main putative recognition sites (named access and deep binding pockets) of MexB and MexY. In this work, we expand the comparison by performing extended molecular dynamics (MD) simulations of these transporters and the pathologically relevant transporter MexF. We employed a more realistic model of the inner phospholipid membrane of P. aeruginosa and more accurate force-fields. To elucidate structure/dynamics-activity relationships we performed physico-chemical analyses and mapped the binding propensities of several organic probes on all transporters. Our data revealed the presence, also in MexF, of a few multifunctional sites at locations equivalent to the access and deep binding pockets detected in MexB. Furthermore, we report for the first time about the multidrug binding abilities of two out of five gates of the channels deputed to peripheral (early) recognition of substrates. Overall, our findings help to define a common “recognition topology” characterizing Mex transporters, which can be exploited to optimize transport and inhibition propensities of antimicrobial compounds.

The increasing interest in the molecular mechanism of the binding of different agonists and antagonists to β

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Fluorescent probes are widely employed to label lipids for the investigation of structural and dynamic properties of model and cell membranes through optical microscopy techniques. Although the effect of tagging a lipid with an organic dye is generally assumed to be negligible, optically modified lipids can nonetheless affect the local lipid structure and, in turn, the lipid lateral mobility. To better assess this potential issue, all-atom (MD) molecular dynamics simulations have been performed to study structural and dynamic effects in a model DOPC membrane in the presence of a standard Rhodamine B-labeled DOPE lipid (RHB) as a function of temperature, i.e., 293K, 303K, and 320K. As the temperature is increased, we observe similar changes in the structural properties of both pure DOPC and RHB-DOPC lipid bilayers: an increase of the area per lipid, a reduction of the membrane thickness and a decrease of lipid order parameters. The partial density profile of the RHB headgroups and their orientation within the lipid bilayer confirm the amphiphilic nature of the RHB fluorescent moiety, which mainly partitions in the DOPC glycerol backbone region at each temperature. Moreover, at all temperatures, our results on lipid lateral diffusion support a non-neutral role of the dye with respect to the unlabeled lipid mobility, thus suggesting important implications for optical microscopy studies of lipid membranes.

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β(2)-adrenergic receptor (β(2)AR). The 3.2 Å x-ray crystal structure of human β(2)AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β(2)AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β(2)AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β(2)AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β(2)AR-ligand complexes performed here.

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD‐EPR simulation approach is two‐fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up‐to‐date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention.

Antimicrobial peptides (AMPs) are small cationic proteins that are able to destabilize a lipid bilayer structure through one or more modes of action. In this study, we investigate the processes of peptide aggregation and pore formation in lipid bilayers and vesicles by the highly cationic AMP, Chrysophsin-3 (chrys-3), using coarse-grained molecular dynamics (CG-MD) simulations and potential of mean force calculations. We study long 50 μs simulations of chrys-3 at different concentrations, both at the surface of dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC) bilayers, and also interacting within the interior of the lipid membrane. We show that aggregation of peptides at the surface, leads to pronounced deformation of lipid bilayers, leading in turn to lipid protrusions for peptide : ligand ratios1 : 12. In addition, aggregation of chrys-3 peptides within the centre of a lipid bilayer leads to spontaneous formation of pores and aggregates. Both mechanisms of interaction are consistent with previously reported experimental data for chrys-3. Similar results are observed also in POPC vesicles and mixed lipid bilayers composed of the zwitterionic lipid palmitoyloleoylphosphatidylethanolamine (POPE) and the negatively charged lipid palmitoyloleoylphosphatidylglycerol (POPG). The latter are employed as models of the bacterial membrane of Escherichia coli.

Summary Biogenesis of high-density lipoproteins (HDL) is coupled to the transmembrane protein, ATP-binding cassette transporter A1 (ABCA1), which transports phospholipid (PL) from the inner to the outer membrane monolayer. Using a combination of computational and experimental approaches, we show that increased outer lipid monolayer surface density, driven by excess PL or membrane insertion of amphipathic helices, results in pleating of the outer monolayer to form membrane-attached discoidal bilayers. Apolipoprotein (apo)A-I accelerates and stabilizes the pleats. In the absence of apoA-I, pleats collapse to form vesicles. These results mimic cells overexpressing ABCA1 that, in the absence of apoA-I, form and release vesicles. We conclude that the basic driving force for nascent discoidal HDL assembly is a PL pump-induced surface density increase that produces lipid monolayer pleating. We then argue that ABCA1 forms an extracellular reservoir containing an isolated pressurized lipid monolayer decoupled from the transbilayer density buffering of cholesterol.

Since spheroidal HDL particles (sHDL) are highly dynamic, molecular dynamics (MD) simulations are useful for obtaining structural models. Here we use MD to simulate sHDL with stoichiometries of reconstituted and circulating particles. The hydrophobic effect during simulations rapidly remodels discoidal HDL containing mixed lipids to sHDL containing a cholesteryl ester/triglyceride (CE/TG) core. We compare the results of simulations of previously characterized reconstituted sHDL particles containing two or three apoA-I created in the absence of phospholipid transfer protein (PLTP) with simulations of circulating human HDL containing two or three apoA-I without apoA-II. We find that circulating sHDL compared with reconstituted sHDL with the same number of apoA-I per particle contain approximately equal volumes of core lipid but significantly less surface lipid monolayers. We conclude that in vitro reconstituted sHDL particles contain kinetically trapped excess phospholipid and are less than ideal models for circulating sHDL particles. In the circulation, phospholipid transfer via PLTP decreases the ratio of phospholipid to apolipoprotein for all sHDL particles. Further, sHDL containing two or three apoA-I adapt to changes in surface area by condensation of common conformational motifs. These results represent an important step toward resolving the complicated issue of the protein and lipid stoichiometry of circulating HDL.

Apolipoprotein (apo) A-I-containing lipoproteins in the form of high-density lipoproteins (HDL) are inversely correlated with atherosclerosis. Because HDL is a soft form of condensed matter easily deformable by thermal fluctuations, the molecular mechanisms for HDL remodeling are not well understood. A promising approach to understanding HDL structure and dynamics is molecular dynamics (MD). In the present study, two computational strategies, MD simulated annealing (MDSA) and MD temperature jump, were combined with experimental particle reconstitution to explore molecular mechanisms for phospholipid- (PL-) rich HDL particle remodeling. The N-terminal domains of full-length apoA-I were shown to be "sticky", acting as a molecular latch largely driven by salt bridges, until, at a critical threshold of particle size, the associated domains released to expose extensive hydrocarbon regions of the PL to solvent. The "sticky" N-termini also associate with other apoA-I domains, perhaps being involved in N-terminal loops suggested by other laboratories. Alternatively, the overlapping helix 10 C-terminal domains of apoA-I were observed to be extremely mobile or "promiscuous", transiently exposing limited hydrocarbon regions of PL. Based upon these models and reconstitution studies, we propose that separation of the N-terminal domains, as particles exceed a critical size, triggers fusion between particles or between particles and membranes, while the C-terminal domains of apoA-I drive the exchange of polar lipids down concentration gradients between particles. This hypothesis has significant biological relevance since lipid exchange and particle remodeling are critically important processes during metabolism of HDL particles at every step in the antiatherogenic process of reverse cholesterol transport.

For several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle ∼100 A in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 36605-36619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28-34-A intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape.

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.

The product of transesterification of phospholipid acyl chains and unesterified cholesterol (UC) by the enzyme lecithin:cholesterol acyltransferase (LCAT) is cholesteryl ester (CE). Activation of LCAT by apolipoprotein (apo) A-I on nascent (discoidal) high-density lipoproteins (HDL) is essential for formation of mature (spheroidal) HDL during the antiatherogenic process of reverse cholesterol transport. Here we report all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of HDL particles that have major implications for mechanisms of LCAT activation. Both the all-atom and CG simulations provide support for a model in which the helix 5/5 domains of apoA-I create an amphipathic "presentation tunnel" that exposes methyl ends of acyl chains at the bilayer center to solvent. Further, CG simulations show that UC also becomes inserted with high efficiency into the amphipathic presentation tunnel with its hydroxyl moiety (UC-OH) exposed to solvent; these results are consistent with trajectory analyses of the all-atom simulations showing that UC is being concentrated in the vicinity of the presentation tunnel. Finally, consistent with known product inhibition of CE-rich HDL by CE, CG simulations of CE-rich spheroidal HDL indicate partial blockage of the amphipathic presentation tunnel by CE. These results lead us to propose the following working hypothesis. After attachment of LCAT to discoidal HDL, the helix 5/5 domains in apoA-I form amphipathic presentation tunnels for migration of hydrophobic acyl chains and amphipathic UC from the bilayer to the phospholipase A2-like and esterification active sites of LCAT, respectively. This hypothesis is currently being tested by site-directed mutagenesis.

Apolipoprotein (apo) A-I is an unusually flexible protein whose lipid-associated structure is poorly understood. Thermal denaturation, which is used to measure the global helix stability of high-density lipoprotein (HDL)-associated apoA-I, provides no information about local helix stability. Here we report the use of temperature jump molecular dynamics (MD) simulations to scan the per-residue helix stability of apoA-I in phospholipid-rich HDL. When three 20 ns MD simulations were performed at 500 K on each of two particles created by MD simulations at 310 K, bilayers remained intact but expanded by 40%, and total apoA-I helicity decreased from 95% to 72%. Of significance, the conformations of the overlapping N- and C-terminal domains of apoA-I in the particles were unusually mobile, exposing hydrocarbon regions of the phospholipid to solvent; a lack of buried interhelical salt bridges in the terminal domains correlated with increased mobility. Nondenaturing gradient gels show that 40% expansion of the phospholipid surface of 100:2 particles by addition of palmitoyloleoylphosphatidylcholine exceeds the threshold of particle stability. As a unifying hypothesis, we propose that the terminal domains of apoA-I are phospholipid concentration-sensitive molecular triggers for fusion/remodeling of HDL particles. Since HDL remodeling is necessary for cholesterol transport, our model for remodeling has substantial biomedical implications.

A combined use of (31)P, (23)Na, (2)H and (17)O NMR spectroscopies and polarized light microscopy has been employed to investigate the effect of the ethidium bromide (EB) binding on the liquid crystalline phase of concentrated double stranded DNA solutions. The optical textures and the (31)P and (23)Na NMR spectra of the DNA anisotropic solutions show that the intercalation of EB induces significant modifications either in the arrangements of the DNA rods and the surrounding ionic atmosphere. On the contrary, no indication of significant changes of the orientational order of the water molecules around DNA emerges from the water (2)H and (17)O NMR spectra.

The interaction of the divalent metal ions Mg(2+), Cd(2+), and Ni(2+) with liquid crystalline NaDNA solutions (molar ratios Me(2+)/DNA-phosphate/=0.050) was investigated by polarized light microscopy and multinuclear (31)P, (2)H, and (23)Na NMR. Our findings show that the state of the cholesteric NaDNA phase at equal MgCl(2), CdCl(2), or NiCl(2) concentration is affected in a different way and to a different extent by the nature of the divalent cation. Indeed, we found that the occurrence of an isotropic phase is markedly favored by Mg(2+), and to a lower extent by Cd(2+), and that the pitch of the cholesteric phase decreases in the presence of Cd(2+) or Ni(2+). (23)Na NMR spectroscopy also shows differences between the binding behavior of Mg(2+) and the transition metal ions in the counterion atmosphere around DNA. The results are discussed in terms of different binding modes of the metal ions.