Putative Transmembrane Helices Research Articles

The role of TMS 12 in the staphylococcal multidrug efflux protein QacA.

To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.

Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition

The RhopH complex is implicated in malaria parasites' ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here, we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1, and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo-electron microscopy (cryo-EM), mass spectrometry, and the cryoID program. CLAG3.1 is positioned between RhopH2 and RhopH3, which both share substantial binding interfaces with CLAG3.1 but make minimal contacts with each other. The forces stabilizing individual subunits include 13 intramolecular disulfide bonds. Notably, CLAG3.1 residues 1210 to 1223, previously predicted to constitute a transmembrane helix, are embedded within a helical bundle formed by residues 979 to 1289 near the C terminus of CLAG3.1. Buried in the core of the RhopH complex and largely shielded from solvent, insertion of this putative transmembrane helix into the erythrocyte membrane would likely require a large conformational rearrangement. Given the unusually high disulfide content of the complex, it is possible that such a rearrangement could be initiated by the breakage of allosteric disulfide bonds, potentially triggered by interactions at the erythrocyte membrane. This first direct observation of an exported Plasmodium falciparum transmembrane protein-in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices-offers insights into the assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential the endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.

Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.

NMR Study of Rcf2 Reveals an Unusual Dimeric Topology in Detergent Micelles.

The Saccharomyces cerevisiae mitochondrial respiratory supercomplex factor 2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochrome bc1 (complex III) and cytochrome c oxidase (complex IV). We expressed the Rcf2 protein in Escherichia coli, refolded it, and reconstituted it into dodecylphosphocholine (DPC) micelles. The structural properties of Rcf2 were studied by solution NMR, and near complete backbone assignment of Rcf2 was achieved. The secondary structure of Rcf2 contains seven helices, of which five are putative transmembrane (TM) helices, including, unexpectedly, a region formed by a charged 20-residue helix at the C terminus. Further studies demonstrated that Rcf2 forms a dimer, and the charged TM helix is involved in this dimer formation. Our results provide a basis for understanding the role of this assembly/regulatory factor in supercomplex formation and function.

BtsT, a Novel and Specific Pyruvate/H+ Symporter in Escherichia coli.

The peptide transporter carbon starvation (CstA) family (transporter classification [TC] 2.A.114) belongs to the second largest superfamily of secondary transporters, the amino acid/polyamine/organocation (APC) superfamily. No representative of the CstA family has previously been characterized either biochemically or structurally, but we have now identified the function of one of its members, the transport protein YjiY of Escherichia coli Expression of the yjiY gene is regulated by the LytS-like histidine kinase BtsS, a sensor of extracellular pyruvate, together with the LytTR-like response regulator BtsR. YjiY consists of 716 amino acids, which form 18 putative transmembrane helices. Transport studies with intact cells provided evidence that YjiY is a specific and high-affinity transporter for pyruvate (Km , 16 μM). Furthermore, reconstitution of the purified YjiY into proteoliposomes revealed that YjiY is a pyruvate/H+ symporter. It has long been assumed that E. coli possesses a transporter(s) for pyruvate, but the present study is the first to definitively identify such a protein. Based on its function, we propose to change the name of the uncharacterized gene yjiY to btsT for Brenztraubensäure (the German word for pyruvate) transporter.IMPORTANCE BtsT (formerly known as YjiY) is found in many commensal and pathogenic representatives of the Enterobacteriaceae This study for the first time characterizes a pyruvate transporter in E. coli, BtsT, as a specific pyruvate/H+ symporter. When nutrients are limiting, BtsT takes up pyruvate from the medium, thus enabling it to be used as a carbon source for the growth and survival of E. coli.

Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking

BackgroundHuman cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. MethodsWe compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. ResultsThe protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A′ and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA′ and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. Major conclusion and general significanceThis model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.

Vba4p, a vacuolar membrane protein, is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae.

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.

Bacillus cereus efflux protein BC3310 - a multidrug transporter of the unknown major facilitator family, UMF-2.

Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.

PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

<i>In Silico</i> Analysis of Cellulose Synthase Gene (NcCesA1) in Developing Xylem Tissues of <i>Neolamarckia Cadamba</i>

This study reported the isolation and in silico characterization of full-length cellulose synthase (CesA) cDNA from Neolamarckia cadamba, an important tropical plantation tree species. CesA is a member of processive glycosyltransferases that involved in cellulose biosynthesis of plants. CesA acts as a central catalyst in the generation of plant cell wall biomass or cellulose. It also plays an important role in regulating wood formation. The hypothetical full-length CesA cDNA (NcCesA1; JX134621) was assembled by contig mapping approach using the CesA cDNA sequences from NcdbEST and the amplified 5�-and 3�-RACE PCR sequences. The NcCesA1 cDNA has a length of 3,472 bp with 3,126 bp open reading frame encoding a 1,042 amino acid sequence. The predicted NcCesA1 protein contained N-terminal cysteine rich zinc binding domain, 7 putative Transmembrane Helices (TMH), 4 U-motifs that contain a signature D, D, D, QxxRW motif, an alternating Conserved Region (CR-P) and 2 Hypervariable Regions (HVR). These entire shared domain structures suggest the functional role of NcCesA1 is involved in glycosyltransferation of the secondary cell wall cellulose biosynthesis of N. cadamba. Sequence comparison also revealed the high similarity (90%) among NcCesA1 and PtrCesA2 of Populus tremuloides. This further implies the involvement of NcCesA1 that catalyzes the cellulose biosynthesis of secondary cell wall rather than primary cell wall. This full-length NcCesA1 cDNA can serve as good candidate gene in association genetics study which leads to Gene-Assisted Selection (GAS) in the N. cadamba tree breeding programme. Furthermore, the isolation of new CesA genes from tropical tree genomes is essential for enhancing knowledge of cellulose biosynthesis in trees that has many fundamental and commercial implications.

A novel mutation within a transmembrane helix of the bile salt export pump (BSEP,ABCB11) with delayed development of cirrhosis

The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC-2) or progressive familial intrahepatic cholestasis (PFIC-2). The molecular basis of BSEP-associated liver disease in a sibling pair was characterized by immunostaining, gene sequencing, bile salt analysis and recombinant expression in mammalian cells and yeast for localization and in vitro activity studies respectively. Benign recurrent intrahepatic cholestasis was considered in a brother and sister who both suffered from intermittent cholestasis since childhood. Gene sequencing of ABCB11 identified the novel missense mutation p.G374S, which is localized in the putative sixth transmembrane helix of BSEP. Liver fibrosis was present in the brother at the age of 18 with progression to cirrhosis within 3 years. Immunofluorescence of liver tissue showed clear canalicular BSEP expression; however, biliary concentration of bile salts was drastically reduced. In line with these in vivo findings, HEK293 cells showed regular membrane targeting of human BSEP(G374S), whereas in vitro transport measurements revealed a strongly reduced transport activity. The novel mutation p.G374S impairs transport function without disabling membrane localization of BSEP. While all other known BSEP mutations within transmembrane helices are associated with PFIC-2, the new p.G374S mutation causes a transitional phenotype between BRIC-2 and PFIC-2.

The single C‐terminal helix of human phospholipid scramblase 1 is required for membrane insertion and scrambling activity

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca(2+)-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca(2+) for ∆CTH-hPLSCR1 (K(a) = 115 μM) compared with hPLSCR1 (K(a) = 249 μM). Far UV-CD studies revealed that the CTH peptide adopts α-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca(2+) coordination and also plays an important role in the functional conformation of hPLSCR1.

Production and initial structural characterization of the TM4TM5 helix‐loop‐helix domain of the translocator protein

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix-helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C-terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far-UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n-dodecyl-β-d-maltoside, n-octyl-β-d-glucoside, n-dodecylphosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5-DHPC complex.

Molecular Modeling of Hydrogenase Enzymes for Biofuel Cell Design

Within the BioPac project, we aim to understand the molecular details of hydrogenase function in order to design and improve next generation biofuel cells. Hydrogenases are key enzymes for the enzymatic conversion of molecular hydrogen into protons and electrons, representing a promising replacement of chemical catalysts in fuel cell devices.To obtain a working biofuel cell, we have been studying the Aquifex Aeolicus (Aa) hydrogenase enzyme because of its resistance to oxygen and carbon monoxyde. Moreover, the presence of a putative transmembrane helix and protein-DDM interaction represent two challenging features to efficiently immobilize the protein on the electrode surface.Using classical molecular dynamics simulations in explicit solvent, we compare the properties of the enzyme from Desulfovibrio fructosovorans, a much studied bacterium, with the one from Aa. We focus mainly on surface properties with the aim to optimize immobilisation on an electrode surface. We also analyze differences among both enzymes as they might provide insight into the origin of oxygen resistance. Moreover, REFT simulations have been used to address both the DDM-protein interaction and the transmembrane helix conformational landscape.In the experimental counterpart of the project, a first working fuel cell prototype was realized yielding 300 μW cm−2 power. Insights on the interaction between hydrogenase model and electrode will drive the experimental design of an improved electrode increasing the efficiency of the association and hence of electron transfer.

Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein

Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.

Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

The a subunit of F(1)F(o) (F(1)F(o)-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H(+)-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F(o) a-less F(1)F(o) complex in Escherichia coli membrane vesicles and liposomes. The resulting F(1)F(o) has a H(+)-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F(1)F(o). By using this procedure, we analysed five mutations of F(1)F(o), where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(o) a subunits were successfully incorporated into F(1)F(o), showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F(1)F(o). The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H(+)-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

Monovalent Ion Selectivity of the Homotetrameric Bacterial Na Channel, NaChBac

NaChBac, from Bacillus halodurans, is a homotetrameric Nav channel, with each monomer having 6 putative transmembrane helices. Its selectivity filter appears to be lined by a ring of 4 glutamate residues (E191 from each monomer). We expressed NaChBac channels in HEK293 cells and studied their selectivity to monovalent organic and alkali cations. Reversal potential shifts were determined from whole-cell currents, following substitution of a test cation for extracellular Na+, and relative permeabilities (PX/PNa) were calculated using the Goldman-Hodgkin-Katz equation. Reversal potentials (Erev) were routinely measured from plots of peak INa vs V, but for ions showing small inward currents, values were checked against tail currents, measured immediately after a maximally activating prepulse. Among the alkali cations, only Na+, Li+ and K+ were measurably permeant. Of 14 organic cations, at pH 7.4, only hydrazinium (HZ+) was measurably permeant (PX/PNa, ≈ 0.8, 0.3, 0.7 respectively, for X= Li, K, HZ; <0.1 for Rb and ammonium). In contrast to eukaryotic, 4-domain Nav channels, neither ammonium nor hydroxyl-ammonium (HA+) was measurably permeant. The NaChBac mutant, E191D, showed diminished selectivity, reflected by increased values for PX/PNa. This mutation conserves the charge in the putative selectivity filter ring, but likely enlarges the filter diameter because of the shorter aspartate side chains, thereby reducing the stringent selectivity attained by the WT channel. For E191D channels, ammonium (PNH4/PNa ≈ 0.4), as well as hydroxyl-ammonium and some other small organic ions, showed measurable permeability. The recently determined crystal structure for the related channel, NavAb (Payandeh et al., 2011, Nature 475:353), opens the way, for the first time, to interpret selectivity of a voltage-dependent Na channel in the context of high-resolution structural data. Overall, our results suggest that NaChBac achieves selectivity for Na by a unique molecular strategy.

A Structural Recognition Mechanism Study of MARCH and its Substrate

Ubiquitination, used by all eukaryotic cells to tag proteins for proteasomal degradation, is also involved in membrane protein regulation. The process of ubiquitination is mediated by ubiquitin ligases, such as the MARCH ligases in this study. The MARCH ligases are membrane proteins and play a major immunoregulatory role in cells of the immune system. They recognize their (membrane protein) substrates via transmembrane (TM) interactions_a poorly understood phenomenon. In order to characterize these TM-induced MARCH-substrate interactions, we have performed replica exchange molecular dynamics (REX-MD) simulations in an implicit membrane to model TM structures of MARCH-1 and MARCH-9 based on their TM and loop sequences. Both MARCHs have two putative TM helices (TM1 and TM2) connected by an extracellular loop. The key TM1-TM2 interfacial residues and the relative orientation of the two TMs are identified and presented. We have also determined possible TM interaction modes of known MARCH-substrate by REX-MD simulations of the complex structure, and the results will be presented in terms of the molecular basis of the MARCH-substrate recognition mechanisms.

Preferential Location of Amino Acids Across the Membrane in α-Helices and β-Barrel Membrane Protein Structures

Membrane proteins perform important physiological functions since they act as sensors for extra-cellular stimuli and gates for transport and communication. However, the basic forces and principles that drive membrane protein folding and assembly remain elusive. The key to the thermodynamic stability of membrane proteins is the complex physiochemical environment of the lipid bilayer. At the most basic level the hydrophobic core of the bilayer has been used to develop hydrophobicity scales that can identify putative transmembrane helices in membrane proteins. Recent refinements have utilized the realization that different amino acid types have strong preferences for particular insertion depths along the membrane normal, allowing a more detailed prediction and differentiation of buried, interfacial, and loop segments.However, prediction of beta-barrel membrane protein segments has so far resisted this analysis. This was primarily due to a lack of sufficient high resolution structures. Here we extend a method previously developed and successfully applied to alpha-helical membrane proteins to beta-barrels. We report the distribution of amino acids along the membrane normal for beta-barrels and compare the differences to alpha-helical proteins. For both protein types membrane interfaces are dominated by small residues such as glycine, alanine, and serine. Charged residues displayed non-symmetric distributions with charged residues generally preferring the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Even though there are many similarities the overall distributions are remarkably different between alpha and beta proteins, with beta-barrels displaying a much narrower hydrophobic core. We confirm that hydrophobic residues are the main driving force behind membrane protein insertion, while polar, charged and aromatic residues were found to be important for the correct orientation of the helix inside the membrane.