Membrane-spanning Domains Research Articles

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β- d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.

Isolation and characterization of an ABC-transporter cDNA clone from wheat (Triticum aestivum L.)

The outmost layer of the surface of any land plant is covered with wax, which protects the inner tissues of plants. The Arabidopsis ATP binding cassette (ABC) transporter, Cer5, was found to be an exporter of wax molecules. Full-length wheat tacer5 cDNA, a sequence homologous to cer5, was isolated. Similar to other transporters of the ABCG subfamily, TaCer5 also has a typical basic structure of NBD-MSD (nucleotide-binding folds domain - membrane-spanning domain). Although the identity scores of TaCer5 and other ABCG transporters are not very high, amino acid residues of NBDs and MSDs are highly conserved among them. Tacer5 is a tissue-specific gene. The transcripts of tacer5 were found in leaf tissues and predominantly in stem tissues but were hardly detectable in root tissues. Stress inhibited the expression of tacer5. The expression was significantly decreased by salinity, low temperature, and 48-hour-darkness and also by exogenous phytohormone abscisic acid and salicylic acid, although not significantly. Exposure to polyethylene glycol either increased the expression or decreased it, depending on the duration of exposure. All the above factors also decreased the extent of wax deposited on wheat leaves. It is therefore concluded that transport of wax molecules is affected by environmental factors.

Dimorphisms in the Membrane-Spanning Domain of EPCR Impact Systemic Coagulation

The role of vascular coagulation receptors, particularly modest changes in the levels of these proteins in terms of controlling downstream coagulation factor activation, remains largely unknown. The study reported by Ireland and colleagues in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology 1 demonstrates that a moderately common dimorphism in the endothelial cell protein C receptor (EPCR) influences the levels of markers of ongoing coagulation (Figure). Several of the key components, thrombomodulin and the EPCR, are subject to downregulation by inflammatory mediators that could play a role potentially in local thrombotic events. EPCR is an important regulatory factor in that it binds protein C and activated protein C,2 augments protein C activation by the thrombin-thrombomodulin complex,3 facilitates activated protein C cytoprotective activity,4 binds factor VII and VIIa,5–6 and leads to the internalization and degradation of both factor VII and protein C.7 In addition, the EPCR gene has a relatively common dimorphism in which there is a Ser to …

P12-04. Topology of the C-terminal tail of HIV-1 gp41: Differential exposure of the Kennedy epitope on cell and viral membranes

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the intracytoplasmic domain based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical Kennedy (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virionassociated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned. © 2010 Steckbeck et al.

The somato-dendritic A-type K+ channel complex: a ménage à trois

The somato-dendritic subthreshold-operating A-type K+ current (ISA) plays a crucial role in neuronal excitability. It regulates spike timing and frequency, dampens the back propagation of action potentials into dendrites and has an important role in dendritic signal processing (Hoffman et al., 1997). Hence, any alteration of ISA current density or gating kinetics could then have important consequences on dendritic integration and plasticity. Kv4 channel protein complexes represent the molecular correlate of the ISA current (Birnbaum et al., 2004; Maffie and Rudy, 2008). Studies in heterologous expression systems have shown that Kv4 pore-forming α subunits interact with two different auxiliary subunits: Kv-channel-interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins DPP6 and DPP10 (DPPX). Both auxiliary subunits affect channel gating and trafficking and their co-expression with Kv4 α subunits recapitulates the kinetics and gating properties of native ISA. The discovery of Nadal et al. (2003) showing that DPP6 proteins can be co-purified with Kv4.2 K+ channel proteins from rat cerebellum and suggesting that they might function as auxiliary subunits of the channel complex, opened new interesting perspectives for the role of this structurally-related dipeptidyl aminopeptidase. Indeed, recent studies showed that mutations in the DPP6 gene are associated with human neurological diseases, including amyotrophic lateral sclerosis (ALS), autism and progressive spinal muscular atrophy. DDP6 is a type II transmembrane protein, highly expressed in the nervous system and belonging to the prolyl oligopeptidase family of serine proteases. It consists of a large extracellular C-terminal domain, a single membrane-spanning domain and a short alternatively spliced intracellular N-terminus. DPP6 has a dimeric structure and has lost a catalytic serine residue, suggesting that it is unlikely to function as a dipeptidyl aminopeptidase (Strop et al., 2004). Though biochemical and functional data strongly suggest that DPP6 is an integral component of the Kv4.2 K+ channel complex and functions as an auxiliary subunit, the lack of good quality antibodies prevented high-resolution localization studies in native brain tissue. In the recent issue of Frontiers, Clark et al. (2008) provide a strong argument supporting the view that DPP6 is an integral part of native Kv4.2 channel complexes (ISA) in the brain. They raised antibodies against the whole extracellular domain of the protein, which was recombinantly expressed in baculovirus-infected Sf9 cells. Using this specific tool, they investigated DPP6 distribution in mouse brain and compared it with the distribution of Kv4 proteins (Clark et al., 2008). The three regions most prominently co-labeled with DPP6 and Kv4.2 antibodies were the striatum, the hippocampus and the cerebellar cortex. In the granule cells of the cerebellum, both Kv4.2 and DPP6 proteins were heavily stained in dendrites with a similar anterior–posterior gradient of expression. In line with the high density of ISA current found in dendrites of hippocampal pyramidal CA1 neurons (Hoffman et al., 1997), the dendrites of CA1–CA3 pyramidal cells were also prominently immunostained for both DPP6 and Kv4.2. In the molecular layer of the dentate gyrus, Kv4.2 and DPP6 staining co-localize in the dendrites of dentate granule cells. Thus, this study found remarkable similarities in the regional and cellular patterns of expression of Kv4.2 and DPP6 proteins. Similarities were also found in the dendritic subcellular pattern of expression of both proteins, notably in the CA1–CA3 hippocampal field and in the granule cells of the cerebellum. Such findings strengthen the notion that DPP6 is an integral part of the Kv4 channel complex that underlies the somato-dendritic ISA current in neurons. However, this study further provides intriguing findings showing that one brain structure, the hippocampal mossy fibers, which contain the axons of the granule cells of the dentate gyrus, do not share this pattern of Kv4 and DPP6 co-expression. The hippocampal mossy fiber axons lack Kv4 proteins but are heavily labeled by DPP6 antibodies, particularly in the terminal fields in the stratum lucidum of the CA3 region, thereby suggesting that DPP6 proteins may have additional, Kv4-unrelated functions. Altogether, these recent studies on DPP6(X) proteins raise important issues. Most likely, the Kv4 channel complex contains two dimers of DPP6 proteins. If so, does DPP6 assemble as homodimer or heterodimer and how the KChIP subunits are engaged into the Kv4 channel complex? Are KChIP and DPP6 exerting their effects on Kv4 channel surface expression via different mechanisms? The absence of Kv4 α subunits and the strong expression of DPP6 in the hippocampal mossy fibers indicate that DPP6(X) proteins may have presynaptic functions. It will be important to investigate the possible role of DPP6 in transmitter release as well as in motor neuron function and excitotoxicity, regarded as one of the main processes involved in the pathogenesis of ALS and progressive muscular spinal atrophy. Addressing these challenging issues will highlight the importance of DPP6 in Kv4-related and – unrelated functions.

Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion, Incorporation, and Infectivity

The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

ABC50 Promotes Translation Initiation in Mammalian Cells

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

Molecular models of the open and closed states of the whole human CFTR protein.

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (DeltaF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

A Comprehensive Approach to Identification of Surface-Exposed, Outer Membrane-Spanning Proteins of Leptospira interrogans

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts or infection of accidental hosts, including humans. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in virulence mechanisms of pathogens and the adaptation to various environmental conditions, including those of the mammalian host. Little is known about the surface-exposed OMPs in Leptospira, particularly those with outer membrane-spanning domains. Herein, we describe a comprehensive strategy for identification and characterization of leptospiral transmembrane OMPs. The genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1–130 allowed us to employ the β-barrel prediction programs, PRED-TMBB and TMBETA-NET, to identify potential transmembrane OMPs. Several complementary methods were used to characterize four novel OMPs, designated OmpL36, OmpL37, OmpL47 and OmpL54. In addition to surface immunofluorescence and surface biotinylation, we describe surface proteolysis of intact leptospires as an improved method for determining the surface exposure of leptospiral proteins. Membrane integration was confirmed using techniques for removal of peripheral membrane proteins. We also demonstrate deficiencies in the Triton X-114 fractionation method for assessing the outer membrane localization of transmembrane OMPs. Our results establish a broadly applicable strategy for the elucidation of novel surface-exposed outer membrane-spanning proteins of Leptospira, an essential step in the discovery of potential virulence factors, diagnostic antigens and vaccine candidates.

Substrate-Induced Conformational Change of the Escherichia coli Membrane Insertase YidC

The membrane insertase YidC from Escherichia coli reversibly binds its substrate Pf3 coat protein. The effect of this initial binding process was examined in vitro by fluorescence quenching of the tryptophan (Trp) residues of YidC which are highly sensitive fluorescent probes for changes of the protein's tertiary structure. Membrane-reconstituted (in DOPC or DOPE/DOPG vesicles) as well as detergent-solubilized (C(12)PC) YidC was titrated with a Trp-free Pf3 coat mutant. Quenching of the intrinsic Trp fluorescence after titration indicates a change in the tertiary structure of YidC upon binding to the Pf3 coat substrate. Analysis of the binding curves taken from the fluorescence data yielded values for the dissociation constant (K(D)) in the range of 0.5-1.8 microM. Titration experiments with two Trp mutants reveal that the change in the tertiary structure involves mainly the membrane-spanning domain. The influence of the different environments on the secondary structure of YidC as well as of the YidC large periplasmic domain (P1) was investigated by circular dichroism (CD). The CD data show that the YidC secondary structure changes upon reconstitution into a membrane environment when compared to the detergent-solubilized state. In particular, the P1 domain of YidC is considerably affected by the detergent C(12)PC. This underlines the importance to study conformational changes with membrane-inserted proteins.

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions

Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb 2+ > Cu 2+ > Zn 2+ = Cd 2+ > Ni 2+ > Co 2+. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn 2+-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn 2+. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn 2+-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN(K344E), as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN(K344E) had lost alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid

Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding.

The little we know on the structure and machinery of V-ATPase

The life of every eukaryotic cell depends on the function of vacuolar H(+)-ATPase (V-ATPase). Today we know that V-ATPase is vital for many more physiological and biochemical processes than it was expected three decades ago when the enzyme was discovered. These range from a crucial role in the function of internal organelles such as vacuoles, lysosomes, synaptic vesicles, endosomes, secretory granules and the Golgi apparatus to the plasma membrane of several organisms and specific tissues, and specialized cells. The overall structure and mechanism of action of the V-ATPase is supposed to be similar to that of the well-characterized F-type ATP synthase (F-ATPase). Both consist of a soluble catalytic domain (V(1) or F(1)) that is coupled to a membrane-spanning domain (V(o) or F(o)) by one or more 'stalk' components. Owing to the complexity and challenging properties of V-ATPase its study is lagging behind that of its relative F-ATPase. Time will tell whether V-ATPase shares an identical mechanism of action with F-ATPase or its mode of operation is unique.

Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.

Intersubunit Interactions Modulate pH-Induced Activation of Membrane Fusion by the Junín Virus Envelope Glycoprotein GPC

The mature arenavirus envelope glycoprotein GPC is a tripartite complex comprising a stable signal peptide (SSP) in addition to the receptor-binding (G1) and transmembrane fusion (G2) subunits. We have shown previously that SSP is a key element in GPC-mediated membrane fusion, and that GPC sensitivity to acidic pH is modulated in part through the lysine residue at position 33 in the ectodomain loop of SSP (J. York and J. H. Nunberg, J. Virol. 80:7775-7780, 2006). A glutamine substitution at this position stabilizes the native GPC complex and thereby prevents the induction of pH-dependent membrane fusion. In efforts to identify the intersubunit interactions of K33, we performed alanine-scanning mutagenesis at charged residues in the membrane-proximal ectodomain of G2 and determined the ability of these mutations to rescue the fusion deficiency in K33Q GPC. Four second-site mutations that specifically complement K33Q were identified (D400A, E410A, R414A, and K417A). Moreover, complementation was also observed at three hydrophobic positions in the membrane-spanning domain of G2 (F427, W428, and F438). Interestingly, all of the complementing mutations restored wild-type pH sensitivity to the K33Q mutant, while none themselves affected the pH of membrane fusion. Our studies demonstrate a specific interaction between SSP and G2 that is involved in priming the native GPC complex for pH-induced membrane fusion. Importantly, this pH-dependent interaction has been shown to be vulnerable to small-molecule compounds that stabilize the native complex and prevent the activation of membrane fusion. A detailed mechanistic understanding of the control of GPC-mediated membrane fusion will be important in guiding the development of effective therapeutics against arenaviral hemorrhagic fever.

Phenylalanine 508 Forms Intra-domain Contact Crucial To CFTR Folding And Dynamics

Cystic fibrosis (CF) is the most prevalent genetically inherited lethal disease in the United States, with an especially high incidence rate among people of European descent and affects at least one in every 2500-4000 newborns. In 90% of CF patients, the disease is caused by the deletion of phenylalanine-508 (Phe508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family. Prior experimental studies suggest that the Phe508 deletion cause the aberrant folding of NBD1 and the misassembly of various CFTR domains. To gain a fuller understanding of the role of Phe508 in channel function and of the impact of its deletion, we constructed a 3D structural model of CFTR using the high-resolution structure of another ABC transporter, Sav1866. The CFTR model predicts, and we validated experimentally, that Phe508 mediates an interaction between NBD1 and the fourth cytoplasmic loop in the second membrane-spanning domain (MSD2). This interface explains the observed sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as in NBD1. We also confirmed other intra-domain contacts between the cytoplasmic and membrane-spanning domains that were predicted by the CFTR model. Furthermore, our electrophysiological measurements and molecular dynamics simulations show that these interfaces regulate channel gating and are highly dynamic. Aside from advancing understanding of CFTR structure and function, this study identifies the region of CFTR that should be targeted in treating cystic fibrosis.

Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.