Residues In Transmembrane Helices Research Articles

Isolation and Preliminary Characterization of Amino Acid Substitution Mutations That Increase the Activity of the Osmoregulated ProP Protein ofSalmonella EntericaSerovar Typhimurium

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.

Characterization of heme‐binding properties of Paracoccus denitrificans Surf1 proteins

Biogenesis of cytochrome c oxidase (COX) is a highly complex process involving >30 chaperones in eukaryotes; those required for the incorporation of the copper and heme cofactors are also conserved in bacteria. Surf1, associated with heme a insertion and with Leigh syndrome if defective in humans, is present as two homologs in the soil bacterium Paracoccus denitrificans, Surf1c and Surf1q. In an in vitro interaction assay, the heme a transfer from purified heme a synthase, CtaA, to Surf1c was followed, and both Surf proteins were tested for their heme a binding properties. Mutation of four strictly conserved amino acid residues within the transmembrane part of each Surf1 protein confirmed their requirement for heme binding. Interestingly the mutation of a tryptophan residue in transmembrane helix II (W200 in Surf1c and W209 in Surf1q) led to a drastic switch in the heme composition, with Surf1 now being populated mostly by heme o, the intermediate in the heme a biosynthetic pathway. This tryptophan residue discriminates between the two heme moieties, apparently coordinates the formyl group of heme a, and most likely presents the cofactor in a spatial orientation suitable for optimal transfer to its target site within subunit I of cytochrome c oxidase.

Pore-opening mechanism in trimeric P2X receptor channels

The opening of ion channels in response to ligand binding, voltage or membrane stretch underlies electrical and chemical signalling throughout biology. Two structural classes of pore-opening mechanisms have been established, including bending of pore-lining helices in the case of tetrameric cation channels, or tilting of such helices in mechanosensitive channels. In this paper, we explore how the structure of the pore changes during opening in P2X receptors by measuring the modification of introduced cysteine residues in transmembrane helices by thiol-reactive reagents, and by engineering metal bridges. Our results are consistent with the X-ray structure of the closed state, and demonstrate that expansion of the gate region in the external pore is accompanied by a significant narrowing of the inner pore, indicating that pore-forming helices straighten on ATP binding to open the channel. This unique pore-opening mechanism has fundamental implications for the role of subunit interfaces in the gating mechanism of P2X receptors and points to a role of the internal pore in ion permeation.

Evidence that Interaction between Conserved Residues in Transmembrane Helices 2, 3, and 7 Are Crucial for Human VPAC1 Receptor Activation

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.

Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3

Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin. Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated. Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V(max) normalized to cell surface protein expression for Lys41>Ala (wild type: 190 +/- 8, Lys41>Ala:16 +/- 4 pmol (mg protein)(-1) min(-1), P < 0.001), whereas V(max) of Lys41>Arg and Arg580>Lys (103 +/- 8 and 123 +/- 14 pmol (mg protein)(-1) min(-1), P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala. We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.

A Novel Multilevel Statistical Method for the Study of the Relationships between Multireceptorial Binding Affinity Profiles and In Vivo Endpoints

The present work introduces a novel method for drug research based on the sequential building of linked multivariate statistical models, each one introducing a different level of drug description. The use of multivariate methods allows us to overcome the traditional one-target assumption and to link in vivo endpoints with drug binding profiles, involving multiple receptors. The method starts with a set of drugs, for which in vivo or clinical observations and binding affinities for potentially relevant receptors are known, and allows obtaining predictions of the in vivo endpoints highlighting the most influential receptors. Moreover, provided that the structure of the receptor binding sites is known (experimentally or by homology modeling), the proposed method also highlights receptor regions and ligand-receptor interactions that are more likely to be linked to the in vivo endpoints, which is information of high interest for the design of novel compounds. The method is illustrated by a practical application dealing with the study of the metabolic side effects of antipsychotic drugs. Herein, the method detects related receptors confirmed by experimental results. Moreover, the use of structural models of the receptor binding sites allows identifying regions and ligand-receptor interactions that are involved in the discrimination between antipsychotic drugs that show metabolic side effects and those that do not. The structural results suggest that the topology of a hydrophobic sandwich involving residues in transmembrane helices (TM) 3, 5, and 6, as well as the assembling of polar residues in TM5, are important discriminators between target/antitarget receptors. Ultimately, this will provide useful information for the design of safer compounds inducing fewer side effects.

Polar Residues in Transmembrane Helices can Dramatically Reduce Mobility on SDS Gels WITHOUT Dimerization

Polar residues in membrane-spanning helices are known to drive oligomerization in biological and synthetic membranes, as well as in membrane mimetic systems such as detergents. Here we report a study of a hydrophobic peptide that contains either an asparagine (N) or a leucine (L) residue in the 12th position of an otherwise hydrophobic segment of 20 amino acids. These peptides are fully alpha-helical in detergents. In SDS polyacrylamide gels, the L12 peptides migrated as monomers while the N12 peptides always migrated as single bands at twice their apparent molecular weights. In sharp contrast, Forster resonance energy transfer (FRET) experiments in SDS showed little evidence of dimerization of N12 under any conditions studied. Experiments were done with labeled peptides at concentrations up to 50 micromolar and at acceptor to donor ratios from 1:1 to 10:1. SDS concentrations ranged from 3.5 to 70 mM. We also performed in situ FRET experiments on the peptide bands in polyacrylamide gels, where there was little excess FRET observed for the slower N12 bands relative to L12 bands. We conclude that N12 is always monomeric in SDS gels, despite the fact that it appears to migrate as a dimer. Dynamic light scattering experiments showed a significant difference between L12/SDS micelles and N12/SDS micelles. We hypothesize that the polar residue in the center of the otherwise hydrophobic helix alters the interactions between the peptides and detergent and that physical differences in the peptide-detergent micelles, such as shape and stoichiometry are responsible for the altered migration of the N12 peptides relative to the L12 peptides.

Helix XI contributes to the entrance of the serotonin transporter permeation pathway

The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP(+) transport. We examined the importance of TMH XI residues F551 and F556 for MPP(+) translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP(+) transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP(+) transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP(+) was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT(Aa)) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.

Cysteine Scanning Mutagenesis and Disulfide Mapping Studies of the TatA Component of the Bacterial Twin Arginine Translocase

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10-16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Regulation of G protein‐coupled cAMP receptor activation by a hydrophobic residue in transmembrane helix 3

cAR1, a G protein-coupled cAMP receptor, is essential for multicellular development of Dictyostelium. We previously identified a cAR1-Ile(104) mutant that appeared to be constitutively activated based on its constitutive phosphorylation, elevated affinity for cAMP, and dominant-negative effects on development as well as specific cAR1 pathways that are subject to adaptation. To investigate how Ile(104) might regulate cAR1 activation, we assessed the consequences of substituting it with all other amino acids. Constitutive phosphorylation of these Ile(104) mutants varied broadly, suggesting that they are activated to varying extents, and was correlated with polarity of the substituting amino acid residue. Remarkably, all Ile(104) substitutions, except for the most conservative, dramatically elevated the receptor's cAMP affinity. However, only a third of the mutants (those with the most polar substitutions) blocked development. These findings are consistent with a model in which polar Ile(104) substitutions perturb the equilibrium between inactive and active cAR1 conformations in favour of the latter. Based on homology with rhodopsin, Ile(104) is likely buried within inactive cAR1 and exposed to the cytoplasm upon activation. We propose that the hydrophobic effect normally promotes burial of Ile(104) and hence cAR1 inactivation, while polar substitution of Ile(104) mitigates this effect, resulting in activation.

Systematic Analysis of the Entire Second Extracellular Loop of the V1a Vasopressin Receptor: KEY RESIDUES, CONSERVED THROUGHOUT A G-PROTEIN-COUPLED RECEPTOR FAMILY, IDENTIFIED

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V(1a) vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

Aqueous Access Pathways in ATP Synthase Subunit a: REACTIVITY OF CYSTEINE SUBSTITUTED INTO TRANSMEMBRANE HELICES 1, 3, AND 5

Subunit a is thought to play a key role in H+ transport-driven rotation of the subunit c ring in Escherichia coli F1F0 ATP synthase. In the membrane-traversing F0 sector of the enzyme, H+ binding and release occurs at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access to surfaces of TMHs 2, 4, and 5 was previously suggested based upon the chemical reactivity of cysteine residues substituted into these helices. Here we have substituted Cys into TMH1 and TMH3 and extended the substitutions in TMH5 to the cytoplasmic surface. One region of TMH3 proved to be moderately Ag+-sensitive and may connect with the Ag+-sensitive region found previously on the periplasmic side of TMH2. A single Cys substitution in TMH1 proved to be both N-ethylmaleimide (NEM)-sensitive and Ag+-sensitive and suggests a possible packing interaction of TMH1 with TMH2 and TMH3. New Ag+- and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM- and Ag+-sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F0 to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

Structure/Function Relationships of CCR8 Agonists and Antagonists: AMINO-TERMINAL EXTENSION OF CCL1 BY A SINGLE AMINO ACID GENERATES A PARTIAL AGONIST

We describe here the interactions of CCR8 with its ligands using both CCR8 transfectants and a T-cell line expressing the receptor endogenously. Of the CCR8 agonists reported previously, only CCL1 and vMIP-I exhibited potency in assays of intracellular calcium flux, chemotaxis, and receptor internalization, this latter mechanism being dependent upon the expression of beta-arrestins 1 and 2 but independent of Galpha(i) signaling. NH(2)-terminal extension of the mature CCL1 sequence by a serine residue (Ser-CCL1) resulted in a partial agonist with a reduced affinity for CCR8, suggesting that the NH(2) terminus of the ligand plays a role in ligand binding to an intrahelical site. Attempts to identify key residues within this site revealed that the conserved glutamic acid residue in transmembrane helix 7, Glu-286, is crucial for trafficking of the receptor to the cell surface, while Asp-97 of transmembrane helix 2 is dispensable. CCL7 was found to inhibit both Ser-CCL1 and vMIP-I responses but not those of CCL1 itself. Similarly, vMIP-I responses were more than 2 orders of magnitude more sensitive to the specific CCR8 antagonist MC148 than those induced by CCL1, which is difficult to reconcile with the reported affinities for the receptor. Collectively, these data suggest that the CCR8 ligands are allotropic, binding to distinct sites within CCR8 and that the human immune system may have evolved to use CCL7 as a selective antagonist of viral chemokine activity at CCR8 but not those of the host ligand.

Cross-linking between Helices within Subunit a of Escherichia coli ATP Synthase Defines the Transmembrane Packing of a Four-helix Bundle

Subunit a of F(1)F(0) ATP synthase is required in the H(+) transport driven rotation of the c-ring of F(0), the rotation of which is coupled to ATP synthesis in F(1). The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu(+2)-catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu(+2), was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.

Interactions between Conserved Residues in Transmembrane Helices 2 and 7 during Angiotensin AT1 Receptor Activation

Site-directed mutagenesis studies and independent molecular modeling studies were combined to investigate the network of inter-residue interactions within the transmembrane region of the angiotensin AT(1a) receptor. Site-directed mutagenesis was focused on residues Tyr292, Asn294, Asn295, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT(1a) receptor activation. Replacement of Tyr292 by aspartate in helix 7 abolished radioligand binding to both Y292D and D74Y/Y292D mutant receptors. Reciprocal mutations of Asp74/Asn294, Ser115/Asn294, Ser252/Asn294, and Asn298/Sen115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT(1a) receptor activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the TM region of the AT(1) receptor and the mutants as well as of their complexes with angiotensin II, which were used to rationalize the possible reasons of impairing of function of some mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct strong interaction in the resting conformation of the AT(1a) receptor. Balance of interactions between residues in helix 2 (as D74) and helix 7 (as N294, N295 and N298) in the AT(1) receptors, however, has a crucial role both in determining their functional activity and levels of their expression.

Threonine-978 in the Transmembrane Segment of the Multidrug Efflux Pump AcrB of Escherichia coli Is Crucial for Drug Transport as a Probable Component of the Proton Relay Network

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals and expels them from cells. It is a proton antiporter belonging to the resistance-nodulation-division (RND) superfamily. Asp407, Asp408, Lys940, and Arg971 in transmembrane (TM) helices of this transporter have been identified as essential amino acid residues that probably function as components of the proton relay system. In this study, we identified a novel residue in TM helix 11, Thr978, as an essential residue by alanine scanning mutagenesis. Its location close to Asp407 suggests that it is also a component of the proton translocation pathway, a prediction confirmed by the similar conformations adopted by T978A, D407A, D408A, and K940A mutant proteins (see the accompanying paper). Sequence alignment of 566 RND transporters showed that this threonine residue is conserved in about 96% of cases. Our results suggest the hypotheses that Thr978 functions through hydrogen bonding with Asp407 and that protonation of the latter alters the salt bridging and hydrogen bonding pattern in the proton relay network, thus initiating a series of conformational changes that ultimately result in drug extrusion.

Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

Succinate dehydrogenase functioning by a reverse redox loop mechanism and fumarate reductase in sulphate-reducing bacteria

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.