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1.  The Human Transporter Associated with Antigen Processing 
The Journal of Biological Chemistry  2012;287(33):28099-28111.
Background: The transporter associated with antigen processing (TAP) is an ABC transporter whose experimental structure is not known.
Results: We have modeled TAP based on the crystal structures of three related ABC transporters.
Conclusion: We identified a possible peptide binding conformation in the transport cycle of TAP.
Significance: These models help interpret experimental data and give information about the transport cycle of ABC transporters in general.
The human transporter associated with antigen processing (TAP) is a member of the ATP binding cassette (ABC) transporter superfamily. TAP plays an essential role in the antigen presentation pathway by translocating cytosolic peptides derived from proteasomal degradation into the endoplasmic reticulum lumen. Here, the peptides are loaded into major histocompatibility class I molecules to be in turn exposed at the cell surface for recognition by T-cells. TAP is a heterodimer formed by the association of two half-transporters, TAP1 and TAP2, with a typical ABC transporter core that consists of two nucleotide binding domains and two transmembrane domains. Despite the availability of biological data, a full understanding of the mechanism of action of TAP is limited by the absence of experimental structures of the full-length transporter. Here, we present homology models of TAP built on the crystal structures of P-glycoprotein, ABCB10, and Sav1866. The models represent the transporter in inward- and outward-facing conformations that could represent initial and final states of the transport cycle, respectively. We described conserved regions in the endoplasmic reticulum-facing loops with a role in the opening and closing of the cavity. We also identified conserved π-stacking interactions in the cytosolic part of the transmembrane domains that could explain the experimental data available for TAP1-Phe-265. Electrostatic potential calculations gave structural insights into the role of residues involved in peptide binding, such as TAP1-Val-288, TAP2-Cys-213, TAP2-Met-218. Moreover, these calculations identified additional residues potentially involved in peptide binding, in turn verified with replica exchange simulations performed on a peptide bound to the inward-facing models.
doi:10.1074/jbc.M112.381251
PMCID: PMC3431710  PMID: 22700967
ABC Transporter; Molecular Docking; Molecular Dynamics; Molecular Modeling; Multidrug Transporters; Peptide Binding; Transporter Associated with Antigen Processing
2.  Docking of μ-Conotoxin GIIIA in the Sodium Channel Outer Vestibule 
Channels (Austin, Tex.)  2007;1(5):344-352.
μ-Conotoxin GIIIA (μ-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na+ channel. The detailed bases for the toxin’s high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. μ-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na+ channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of μ-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.
PMCID: PMC3212855  PMID: 18690041
ion channel; structural biology; mutational analysis; molecular model; toxin; computer simulation
3.  Alamethicin in Lipid Bilayers: Combined use of X-ray Scattering and MD Simulations 
Biochimica et biophysica acta  2009;1788(6):1387-1397.
Summary
We study fully hydrated bilayers of two di-monounsaturated phospholipids diC18:1PC (DOPC) and diC22:1PC with varying amounts of alamethicin (Alm). We combine the use of X-ray diffuse scattering and molecular dynamics simulations to determine the orientation of alamethicin in model lipids. Comparison of the experimental and simulated form factors shows that Alm helices are inserted transmembrane at high humidity and high concentrations, in agreement with earlier results. The x-ray scattering data and the MD simulations agree that membrane thickness changes very little up to 1/10 Alm/DOPC. In contrast, the x-ray data indicate that the thicker diC22:1PC membrane, thins with added Alm, a total decrease in thickness of 4 Å at 1/10 Alm/diC22:1PC. The different effect of Alm on the thickness changes of the two bilayers is consistent with Alm having a hydrophobic thickness close to the hydrophobic thickness of 27 Å for DOPC; Alm is then mismatched with the 7 Å thicker diC22:1PC bilayer. The x-ray data indicate that Alm decreases the bending modulus (KC) by a factor of ~2 in DOPC and a factor of ~10 in diC22:1PC membranes(P/L ~1/10). The van der Waals and fluctuational interactions between bilayers are also evaluated through determination of the anisotropic B compressibility modulus.
doi:10.1016/j.bbamem.2009.02.013
PMCID: PMC2693350  PMID: 19248763
Lipid bilayers; peptides; x-ray; structure; MD simulation; alamethicin
4.  Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins 
European Biophysics Journal   2009;39(2):229-239.
Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, α-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein.
doi:10.1007/s00249-009-0527-9
PMCID: PMC2795155  PMID: 19669748
Membrane proteins; Side-chain conformations; Tryptophan; Energy transfer (FRET); Computer simulation
5.  Transmembrane Helix 12 Modulates Progression of the ATP Catalytic Cycle in ABCB1† 
Biochemistry  2009;48(26):6249-6258.
Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.
doi:10.1021/bi900373x
PMCID: PMC2791873  PMID: 19456124
6.  Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane 
PLoS ONE  2009;4(11):e7966.
Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.
doi:10.1371/journal.pone.0007966
PMCID: PMC2779261  PMID: 19956595
8.  Computer simulation of partitioning of ten pentapeptides Ace-WLXLL at the cyclohexane/water and phospholipid/water interfaces 
BMC Biochemistry  2005;6:30.
Background
Peptide-membrane interactions play a key role in the binding, partitioning and folding of membrane proteins, the activity of antimicrobial and fusion peptides, and a number of other processes. To gain a better understanding of the thermodynamics of such interactions, White and Wimley created an interfacial hydrophobicity scale based of the transfer free energy from water to octanol or lipid bilayers of a series of synthetic peptapeptides (Ace-WLXLL, with X being any of the twenty natural amino acids) (White and Wimley (1996) Nat. Struct. Biol. 3, 842–848). In this study, we performed molecular dynamics simulations of a representative set of ten of these peptides (X = D, K, R, N, A, T, S, I, F and W) in two membrane mimetic interfaces: water-cyclohexane (10 ns) and a fully solvated dioleoylphosphatidylcholine (DOPC) bilayer (50 ns) using both constant pressure and constant area ensembles. We focus on partitioning of the ten peptides at the cyclohexane/water and lipid/water interfaces.
Results
The peptides rapidly equilibrate (< 2 ns) and partition at the cyclohexane/water interface. The X3 guest residue assumes average orientations that depend on the nature of the side chain. At the DOPC/water interface, dynamics is much slower and convergence is difficult to achieve on a 50 ns timescale. Nonetheless, all peptides partition to the lipid/water interface with distributions with widths of 1–2 nm. The peptides assume a broad range of side chain and backbone orientations and have only a small effect on the area of the unit cell. On average, hydrophobic guest residues partition deeper into the hydrophobic core than hydrophilic residues. In some cases the peptides penetrate sufficiently deep to somewhat affect the distribution of the C=C double bond in DOPC. The relative distribution of the X3 guest residue compared to W1 and L5 is similar in the water/cyclohexane and water/lipid simulations. Snapshots show mostly extended backbone conformations in both environments. There is little difference between simulations at a constant area of 0.66 nm2 and simulations at constant pressure that approximately yield the same average area of 0.66 nm2.
Conclusion
These peptides were designed to assume extended conformations, which is confirmed by the simulations. The distribution of the X3 side chain depends on its nature, and can be determined from molecular dynamics simulations. The time scale of peptide motion at a phospholipids-water interface is too long to directly calculate the experimentally measured hydrophobicity scale to test and improve the simulation parameters. This should be possible at the water/cyclohexane interface and likely will become feasible in the future for the phospholipids/water case.
doi:10.1186/1471-2091-6-30
PMCID: PMC1351180  PMID: 16368010
9.  The molecular basis of electroporation 
BMC Biochemistry  2004;5:10.
Background
Electroporation is a common method to introduce foreign molecules into cells, but its molecular basis is poorly understood. Here I investigate the mechanism of pore formation by direct molecular dynamics simulations of phospholipid bilayers of a size of 256 and of more than 2000 lipids as well as simulations of simpler interface systems with applied electric fields of different strengths.
Results
In a bilayer of 26 × 29 nm multiple pores form independently with sizes of up to 10 nm on a time scale of nanoseconds with an applied field of 0.5 V/nm. Pore formation is accompanied by curving of the bilayer. In smaller bilayers of ca. 6 × 6 nm, a single pore forms on a nanosecond time scale in lipid bilayers with applied fields of at least 0.4 V/nm, corresponding to transmembrane voltages of ca. 3 V. The presence of 1 M salt does not seem to change the mechanism. In an even simpler system, consisting of a 3 nm thick octane layer, pores also form, despite the fact that there are no charged headgroups and no salt in this system. In all cases pore formation begins with the formation of single-file like water defects penetrating into the bilayer or octane.
Conclusions
The simulations suggest that pore formation is driven by local electric field gradients at the water/lipid interface. Water molecules move in these field gradients, which increases the probability of water defects penetrating into the bilayer interior. Such water defects cause a further increase in the local electric field, accelerating the process of pore formation. The likelihood of pore formation appears to be increased by local membrane defects involving lipid headgroups. Simulations with and without salt show little difference in the observed pore formation process. The resulting pores are hydrophilic, lined by phospholipid headgroups.
doi:10.1186/1471-2091-5-10
PMCID: PMC489962  PMID: 15260890
10.  Improving Internal Peptide Dynamics in the Coarse-Grained MARTINI Model: Toward Large-Scale Simulations of Amyloid- and Elastin-like Peptides 
We present an extension of the coarse-grained MARTINI model for proteins and apply this extension to amyloid- and elastin-like peptides. Atomistic simulations of tetrapeptides, octapeptides, and longer peptides in solution are used as a reference to parametrize a set of pseudodihedral potentials that describe the internal flexibility of MARTINI peptides. We assess the performance of the resulting model in reproducing various structural properties computed from atomistic trajectories of peptides in water. The addition of new dihedral angle potentials improves agreement with the contact maps computed from atomistic simulations significantly. We also address the question of which parameters derived from atomistic trajectories are transferable between different lengths of peptides. The modified coarse-grained model shows reasonable transferability of parameters for the amyloid- and elastin-like peptides. In addition, the improved coarse-grained model is also applied to investigate the self-assembly of β-sheet forming peptides on the microsecond time scale. The octapeptides SNNFGAIL and (GV)4 are used to examine peptide aggregation in different environments, in water, and at the water–octane interface. At the interface, peptide adsorption occurs rapidly, and peptides spontaneously aggregate in favor of stretched conformers resembling β-strands.
doi:10.1021/ct200876v
PMCID: PMC3348680  PMID: 22582033
11.  Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core 
Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.
doi:10.1021/jp303267y
PMCID: PMC3434764  PMID: 22962627
13.  Association of Lipidome Remodeling in the Adipocyte Membrane with Acquired Obesity in Humans 
PLoS Biology  2011;9(6):e1000623.
The authors describe a new approach to studying cellular lipid profiles and propose a compensatory mechanism that may help maintain the normal membrane function of adipocytes in the context of obesity.
Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels of ethanolamine plasmalogens containing arachidonic acid. Information gathered from these experimental groups was used for molecular dynamics simulations of lipid bilayers combined with dependency network analysis of combined clinical, lipidomics, and gene expression data. The simulations suggested that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We also show by in vitro Elovl6 knockdown that the lipid network regulating the observed remodeling may be amenable to genetic modulation. Together, our novel approach suggests a physiological mechanism by which adaptation of adipocyte membranes to adipose tissue expansion associates with positive energy balance, potentially leading to higher vulnerability to inflammation in acquired obesity. Further studies will be needed to determine the cause of this effect.
Author Summary
Obesity is characterized by excess body fat, which is predominantly stored in the adipose tissue. When adipose tissue expands too much it stops storing lipid appropriately. The excess lipid accumulates in organs such as muscle, liver, and pancreas, causing metabolic disease. In this study, we aim to identify factors that cause adipose tissue to malfunction when it reaches its limit of expansion. We performed lipidomic analyses of human adipose tissue in twin pairs discordant for obesity—that is, one of the twins was lean and one was obese—but still metabolically healthy. We identified multiple changes in membrane phospholipids. Using computer modeling, we show that “lean” and “obese” membrane lipid compositions have the same physical properties despite their different compositions. We hypothesize that this represents allostasis—changes in lipid membrane composition in obesity occur to protect the physical properties of the membranes. However, protective changes cannot occur without a cost, and accordingly we demonstrate that switching to the “obese” lipid composition is associated with higher levels of adipose tissue inflammation. In a separate group of metabolically unhealthy obese individuals we investigated how the processes that regulate the “lean” and “obese” lipid profiles are changed. To determine how these lipid membrane changes are regulated we constructed an in silico network model that identified key control points and potential molecular players. We validated this network by performing genetic manipulations in cell models. Therapeutic targeting of this network may open new opportunities for the prevention or treatment of obesity-related metabolic complications.
doi:10.1371/journal.pbio.1000623
PMCID: PMC3110175  PMID: 21666801

Results 1-13 (13)