Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane beta-barrel containing a central alpha-helix. The role of this alpha-helix, and of the conformational dynamics of the beta-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug'. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Identification of a cation transport pathway in Neisseria meningitidis PorB

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion‐selective, cation translocation through nucleotide‐bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs⁺, which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs⁺ form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

Misfolding of a bacterial autotransporter

The adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated into the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We have previously shown that the beta-domain can be folded from the urea-denatured state when bound to a nickel column during purification. It has not been possible to achieve proper refolding of the beta-domain in solution; instead, a misfolded state C is formed. Here, we characterize this misfolded state in greater detail, showing that despite being misfolded, C can be analyzed as a conventional conformational state, with cooperative unfolding in urea and SDS as well as showing simple exponential kinetics during its formation in the presence of lipid vesicles and detergent micelles. The kinetics of formation of C is sensitive to the lipid composition in vesicles. We have also attempted to identify biological factors that might aid folding of the beta-domain to the properly folded state. However, no purified periplasmic or cytosolic chaperone was found to increase folding yields, and no factor in a periplasmic extract was identified that could bind to C. We conclude that it is the exposure to the unique spatial arrangement of the bacterial cell that leads to proper refolding of the beta-domain.     

Modeling and simulations of a bacterial outer membrane protein: OprF from Pseudomonas aeruginosa

OprF is a major outer membrane protein from Pseudomonas aeruginosa, a homolog of OmpA from Escherichia coli. The N-terminal domains of both proteins have been demonstrated to form low conductance channels in lipid bilayers. Homology models, consisting of an eight-stranded beta-barrel, of the N-terminal domain OprF have been constructed based on the crystal structure of the corresponding domain from E. coli OmpA. OprF homology models have been evaluated via a set (6 x 10 ns) of simulations of the beta-barrel embedded within a solvated dimyristoyl-phosphatidylcholine (DMPC) bilayer. The conformational stability of the models is similar to that of the crystal structure of OmpA in comparable simulations. There is a degree of water penetration into the pore-like center of the OprF barrel. The presence of an acidic/basic (E8/K121) side-chain interaction within the OprF barrel may form a "gate" able to close/open a central pore. Lipid-protein interactions within the simulations were analyzed and revealed that aromatic side-chains (Trp, Tyr) of OprF interact with lipid headgroups. Overall, the behavior of the OprF model in simulations supports the suggestion that this molecule is comparable to OmpA. The simulations help to explain the mechanism of formation of low conductance pores within the outer membrane.     

Molecular dynamics simulations of ribonuclease T1

Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2GMP-RNase T1 complex. The presence of water led to the maintenance of the 2-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of activesite residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2GMP, which did show an increase in mobility. Analysis of the 2GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.Abbreviations RNase T1 Ribonuclease T1 (EC.3.1.27.3) - 2GMP Guanosine-2-monophosphate - SBS Stochastic Bondary Simulation - VS Vacuum Simulation - MD Molecular Dynamics     

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.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

Molecular dynamics simulations of the interaction of phospholipid bilayers with polycaprolactone

The molecular interaction between common polymer chains and the cell membrane is unknown. Molecular dynamics simulations offer an emerging tool to characterise the nature of the interaction between common degradable polymer chains used in biomedical applications, such as polycaprolactone, and model cell membranes. Herein we characterise with all-atomistic and coarse-grained molecular dynamics simulations the interaction between single polycaprolactone chains of varying chain lengths with a phospholipid membrane. We find that the length of the polymer chain greatly affects the nature of interaction with the membrane, as well as the membrane properties. Furthermore, we next utilise advanced sampling techniques in molecular dynamics to characterise the two-dimensional free energy surface for the interaction of varying polymer chain lengths (short, intermediate, and long) with model cell membranes. We find that the free energy minimum shifts from the membrane-water interface to the hydrophobic core of the phospholipid membrane as a function of chain length. Finally, we perform coarse-grained molecular dynamics simulations of slightly larger membranes with polymers of the same length and characterise the results as compared with all-atomistic molecular dynamics simulations. These results can be used to design polymer chain lengths and chemistries to optimise their interaction with cell membranes at the molecular level.     

Response of Neisseria meningitidis to iron limitation

In the human body, the concentration of free iron is limiting for bacterial growth, since iron is bound to transport and storage proteins such as transferrin and lactoferrin. When grown under iron starvation, Neisseria meningitidis produces receptors for these proteins in the outer membrane. These receptors are presently being characterized at the molecular level. Here, we summarize our current knowledge of these receptors, with special emphasis on the LbpA and FrpB proteins, which are studied in our laboratories. Furthermore, the genetic and antigenic variability of these proteins and their vaccine potential are discussed.     

Molecular dynamics simulations of Factor Xa: insight into conformational transition of its binding subsites

Protein flexibility and conformational diversity is well known to be a key characteristic of the function of many proteins. Human blood coagulation proteins have multiple substrates, and various protein-protein interactions are required for the smooth functioning of the coagulation cascade to maintain blood hemostasis. To address how a protein may cope with multiple interactions with its structurally diverse substrates and the accompanied structural changes that may drive these changes, we studied human Factor X. We employed 20 ns of molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on two different conformational forms of Factor X, open and closed, and observed an interchangeable conformational transition from one to another. This work also demonstrates the roles of various aromatic residues involved in aromatic-aromatic interactions, which make this dynamic transition possible.     

In silico predictions of LH2 ring sizes from the crystal structure of a single subunit using molecular dynamics simulations

Most of the currently known light‐harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like “what factors govern the LH2 ring size?” and “are there other ring sizes possible?” remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8‐ring from Rs. moliscianum (MOLI) and a 9‐ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8‐, 9‐, and 10‐ring geometries. After inserting each of the dimers into a lipid‐water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9‐ring, while MOLI is more likely to form an 8‐ring than a 9‐ring. Finally, we discuss both the merits and limitations of all three prediction methods. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Molecular dynamics simulations from putative transition states of alpha-spectrin SH3 domain

A series of molecular dynamics simulations in explicit solvent were started from nine structural models of the transition state of the SH3 domain of alpha-spectrin, which were generated by Lindorff-Larsen et al. (Nat Struct Mol Biol 2004;11:443-449) using molecular dynamics simulations in which experimental Phi - values were incorporated as restraints. Two of the nine models were simulated 10 times for 200 ns and the remaining models simulated two times for 200 ns. Complete folding was observed in one case, while in the other simulations partial folding or unfolding events were observed, which were characterized by a regularization of elements of secondary structure. These results are consistent with recent experimental evidence that the folding of SH3 domains involves low populated intermediate states.     

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.