Influence of Ganglioside GM1 Concentration on Lipid Clustering and Membrane Properties and Curvature.

Gangliosides are a class of glycosphingolipids (GSLs) with amphiphilic character that are found at the outer leaflet of the cell membranes, where their ability to organize into special domains makes them vital cell membrane components. However, a molecular understanding of GSL-rich membranes in terms of their clustered organization, stability, and dynamics is still elusive. To gain molecular insight into the organization and dynamics of GSL-rich membranes, we performed all-atom molecular-dynamics simulations of bicomponent ganglioside GM1 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayers with varying concentrations of GM1 (10%, 20%, and 30%). Overall, the simulations show very good agreement with available experimental data, including x-ray electron density profiles along the membrane normal, NMR carbohydrate proton-proton distances, and x-ray crystal structures. This validates the quality of our model systems for investigating GM1 clustering through an ordered-lipid-cluster analysis. The increase in GM1 concentration induces tighter lipid packing, driven mainly by inter-GM1 carbohydrate-carbohydrate interactions, leading to a greater preference for the positive curvature of GM1-containing membranes and larger cluster sizes of ordered-lipid clusters (with a composite of GM1 and POPC). These clusters tend to segregate and form a large percolated cluster at a 30% GM1 concentration at 293 K. At a higher temperature of 330 K, however, the segregation is not maintained.

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly ß-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein ß-barrel assembly machine (BAM) catalyzes the insertion and folding of the ß-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane ß-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate ß-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane ß-barrel proteins.

Dynamics and Interactions of OmpF and LPS: Influence on Pore Accessibility and Ion Permeability.

The asymmetric outer membrane of Gram-negative bacteria is formed of the inner leaflet with phospholipids and the outer leaflet with lipopolysaccharides (LPS). Outer membrane protein F (OmpF) is a trimeric porin responsible for the passive transport of small molecules across the outer membrane of Escherichia coli. Here, we report the impact of different levels of heterogeneity in LPS environments on the structure and dynamics of OmpF using all-atom molecular dynamics simulations. The simulations provide insight into the flexibility and dynamics of LPS components that are highly dependent on local environments, with lipid A being the most rigid and O-antigen being the most flexible. Increased flexibility of O-antigen polysaccharides is observed in heterogeneous LPS systems, where the adjacent O-antigen repeating units are weakly interacting and thus more dynamic, compared to homogeneous LPS systems in which LPS interacts strongly with each other with limited overall flexibility due to dense packing. The model systems were validated by comparing molecular-level details of interactions between OmpF surface residues and LPS core sugars with experimental data, establishing the importance of LPS core oligosaccharides in shielding OmpF surface epitopes recognized by monoclonal antibodies. There are LPS environmental influences on the movement of bulk ions (K(+) and Cl(-)), but the ion selectivity of OmpF is mainly affected by bulk ion concentration.

Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein.

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with α-helical structure, whereas PrPSc contains a high proportion of ß-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface.

Lipopolysaccharide membrane building and simulation.

While membrane simulations are widely employed to study the structure and dynamics of various lipid bilayers and membrane proteins in the bilayers, simulations of lipopolysaccharides (LPS) in membrane environments have been limited due to their structural complexity, difficulties in building LPS-membrane systems, and lack of the appropriate molecular force fields. In this work, as a first step to extend CHARMM-GUI Membrane Builder to incorporate LPS molecules and to explore their structures and dynamics in membrane environments using molecular dynamics simulations, we describe step-by-step procedures to build LPS bilayer systems using CHARMM and the recently developed CHARMM carbohydrate and lipid force fields. Such procedures are illustrated by building various bilayers of Escherichia coli R1.O6 LPS and the presentation of preliminary simulation results in terms of per-LPS area and density distributions of various components along the membrane normal.

Rare loss-of-function mutation in complement component C3 provides insight into molecular and pathophysiological determinants of complement activity.

The plasma protein C3 is a central element in the activation and effector functions of the complement system. A hereditary dysfunction of C3 that prevents complement activation via the alternative pathway (AP) was described previously in a Swedish family, but its genetic cause and molecular consequences have remained elusive. In this study, we provide these missing links by pinpointing the dysfunction to a point mutation in the ß-chain of C3 (c.1180T > C; p.Met(373)Thr). In the patient's plasma, AP activity was completely abolished and could only be reconstituted with the addition of normal C3. The M373T mutation was localized to the macroglobulin domain 4 of C3, which contains a binding site for the complement inhibitor compstatin and is considered critical for the interaction of C3 with the AP C3 convertase. Structural analyses suggested that the mutation disturbs the integrity of macroglobulin domain 4 and induces conformational changes that propagate into adjacent regions. Indeed, C3 M373T showed an altered binding pattern for compstatin and surface-bound C3b, and the presence of Thr(373) in either the C3 substrate or convertase-affiliated C3b impaired C3 activation and opsonization. In contrast to known gain-of-function mutations in C3, patients affected by this loss-of-function mutation did not develop familial disease, but rather showed diverse and mostly episodic symptoms. Our study therefore reveals the molecular mechanism of a relevant loss-of-function mutation in C3 and provides insight into the function of the C3 convertase, the differential involvement of C3 activity in clinical conditions, and some potential implications of therapeutic complement inhibition.

Lipid-linked oligosaccharides in membranes sample conformations that facilitate binding to oligosaccharyltransferase.

Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs' preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST.

CHARMM-GUI Membrane Builder toward realistic biological membrane simulations.

CHARMM-GUI Membrane Builder, http://www.charmm-gui.org/input/membrane, is a web-based user interface designed to interactively build all-atom protein/membrane or membrane-only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance-based algorithm for faster initial ion displacement, (5) CHARMM inputs for P21 image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM-GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments.

E. coli outer membrane and interactions with OmpLA.

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Šthinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.

Preferred orientations of phosphoinositides in bilayers and their implications in protein recognition mechanisms.

Phosphoinositides (PIPs), phosphorylated derivatives of phosphatidylinositol (PI), are essential regulatory lipids involved in various cellular processes, including signal transduction, membrane trafficking, and cytoskeletal remodeling. To gain insight into the protein-PIPs recognition process, it is necessary to study the inositol ring orientation (with respect to the membrane) of PIPs with different phosphorylation states. In this study, 8 PIPs (3 PIP, 2 PIP2, and 3 PIP3) with different phosphorylation and protonation sites have been separately simulated in two mixed bilayers (one with 20% phosphatidylserine (PS) lipids and another with PS lipids switched to phosphatidylcholine (PC) lipids), which roughly correspond to yeast membranes. Uniformity of the bilayer properties including hydrophobic thickness, acyl chain order parameters, and heavy atom density profiles is observed in both PS-contained and PC-enriched membranes due to the same hydrophobic core composition. The relationship between the inositol ring orientation (tilt and rotation angles) and its solvent-accessible surface area indicates that the orientation is mainly determined by its solvation energy. Different PIPs exhibit a clear preference in the inositol ring rotation angle. Surprisingly, a larger proportion of PIPs inositol rings stay closer to the surface of PS-contained membranes compared to PC-enriched ones. Such a difference is rationalized with the formation of more hydrogen bonds between the PS/PI headgroups and the PIPs inositol rings in PS-contained membranes. This hydrogen bond network could be functionally important; thus, the present results can potentially add important and detailed features into the existing protein-PIPs recognition mechanism.

ST-analyzer: a web-based user interface for simulation trajectory analysis.

Molecular dynamics (MD) simulation has become one of the key tools to obtain deeper insights into biological systems using various levels of descriptions such as all-atom, united-atom, and coarse-grained models. Recent advances in computing resources and MD programs have significantly accelerated the simulation time and thus increased the amount of trajectory data. Although many laboratories routinely perform MD simulations, analyzing MD trajectories is still time consuming and often a difficult task. ST-analyzer, http://im.bioinformatics.ku.edu/st-analyzer, is a standalone graphical user interface (GUI) toolset to perform various trajectory analyses. ST-analyzer has several outstanding features compared to other existing analysis tools: (i) handling various formats of trajectory files from MD programs, such as CHARMM, NAMD, GROMACS, and Amber, (ii) intuitive web-based GUI environment--minimizing administrative load and reducing burdens on the user from adapting new software environments, (iii) platform independent design--working with any existing operating system, (iv) easy integration into job queuing systems--providing options of batch processing either on the cluster or in an interactive mode, and (v) providing independence between foreground GUI and background modules--making it easier to add personal modules or to recycle/integrate pre-existing scripts utilizing other analysis tools. The current ST-analyzer contains nine main analysis modules that together contain 18 options, including density profile, lipid deuterium order parameters, surface area per lipid, and membrane hydrophobic thickness. This article introduces ST-analyzer with its design, implementation, and features, and also illustrates practical analysis of lipid bilayer simulations.

Molecular dynamics and NMR spectroscopy studies of E. coli lipopolysaccharide structure and dynamics.

Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From (1)H,(1)H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.

Novel analogues of the therapeutic complement inhibitor compstatin with significantly improved affinity and potency.

Compstatin is a 13-residue disulfide-bridged peptide that inhibits a key step in the activation of the human complement system. Compstatin and its derivatives have shown great promise for the treatment of many clinical disorders associated with unbalanced complement activity. To obtain more potent compstatin analogues, we have now performed an N-methylation scan of the peptide backbone and amino acid substitutions at position 13. One analogue (Ac-I[CVW(Me)QDW-Sar-AHRC](NMe)I-NH(2)) displayed a 1000-fold increase in both potency (IC(50) = 62 nM) and binding affinity for C3b (K(D) = 2.3 nM) over that of the original compstatin. Biophysical analysis using surface plasmon resonance and isothermal titration calorimetry suggests that the improved binding originates from more favorable free conformation and stronger hydrophobic interactions. This study provides a series of significantly improved drug leads for therapeutic applications in complement-related diseases, and offers new insights into the structure-activity relationships of compstatin analogues.

Coriolis coupling and nonadiabaticity in chemical reaction dynamics.

The nonadiabatic quantum dynamics and Coriolis coupling effect in chemical reaction have been reviewed, with emphasis on recent progress in using the time-dependent wave packet approach to study the Coriolis coupling and nonadiabatic effects, which was done by K. L. Han and his group. Several typical chemical reactions, for example, H+D(2), F+H(2)/D(2)/HD, D(+)+H(2), O+H(2), and He+H(2)(+), have been discussed. One can find that there is a significant role of Coriolis coupling in reaction dynamics for the ion-molecule collisions of D(+)+H(2), Ne+H(2)(+), and He+H(2)(+) in both adiabatic and nonadiabatic context.

Determination of the structure form of the fourth ligand of zinc in Acutolysin A using combined quantum mechanical and molecular mechanical simulation.

Acutolysin A, which is isolated from the snake venom of Agkistrodon acutus, is a member of the SVMPs subfamily of the metzincin family, and it is a snake venom zinc metalloproteinase possessing only one catalytic domain. The catalytic zinc ion, in the active site, is coordinated in a tetrahedral manner with three imidazole nitrogen atoms of histidine and one oxygen atom. It is uncertain whether this oxygen atom is a water molecule or a hydroxide ion just from the three-dimensional X-ray crystal structure. The identity of the fourth ligand of zinc is theoretically determined for the first time by performing both combined quantum mechanical and molecular mechanical (QM/MM) simulation and high-level quantum mechanical calculations. All of the results obtained indicate that the fourth ligand in the active site of the reported X-ray crystal structure is a water molecule rather than a hydroxide anion. On the basis of these theoretical results, we note that the experimental observed pH dependence of the proteolytic and hemorrhagic activity of Acutolysin A can be attributed to the deprotonation of the zinc-bound water to yield a better nucleophile, the hydroxide ion. Structural analyses revealed structural details useful for the understanding of acutolysin catalytic mechanism.

Selectivity of neutral/weakly basic P1 group inhibitors of thrombin and trypsin by a molecular dynamics study.

Molecular dynamics (MD) simulations followed by molecular mechanics generalized Born surface area (MM-GBSA) analyses have been carried out to study the selectivity of two neutral and weakly basic P1 group inhibitors (177 and CDA) to thrombin and trypsin. Detailed binding free energies between these inhibitors and individual protein residues are calculated by using a per-residue basis decomposition method. The analysis of the detailed interaction energies provides insight on the protein-inhibitor-binding mechanism and helps to elucidate the basis for achieving selectivity through interpretation of the structural and energetic results from the simulations. The study shows that the dominant factor of selectivity for both inhibitors is van der Waals energy, which suggests better shape complementarity and packing with thrombin. Nonpolar solvation free energy and total entropy contribution are also in favor of selectivity, but the contributions are much smaller. Binding mode and structural analysis show that 177 binds to thrombin and trypsin in a similar binding mode. In contrast, the CDA binds to thrombin and trypsin in very different modes.

Quantum and molecular dynamics study for binding of macrocyclic inhibitors to human alpha-thrombin.

Molecular dynamics simulations followed by quantum mechanical calculation and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis have been carried out to study binding of proline- and pyrazinone-based macrocyclic inhibitors (L86 and T76) to human alpha-thrombin. Detailed binding interaction energies between these inhibitors and individual protein fragments are calculated using DFT method based on a new quantum mechanical approach for computing protein-ligand interaction energy. The analysis of detailed interaction energies provides insight on the protein-ligand binding mechanism. Study shows that T76 and L86 bind to thrombin in a very similar "inhibition mode" except that T76 has relatively weaker binding interaction with Glu(217). The analysis from quantum calculation of binding interaction is consistent with the MM-PBSA calculation of binding free energy, and the calculated free energies for L86/T76-thrombin binding agree well with the experimental data.