Amino Acids In Transmembrane Domain Research Articles

Variation in the tonoplast cadmium transporter heavy metal ATPase 3 (HMA3) homolog gene in Aegilops tauschii.

The functionality of HMA3 is a key determinant controlling Cd accumulation in the shoots and grains of plants. Wild relatives of modern crop plants can serve as sources of valuable genetic variation for various traits. Here, resequencing of HMA3 homoeologous genes from Aegilops tauschii (the donor of the wheat D genome) was carried out to identify natural variation at both the nucleotide and polypeptide levels. HMA3 homoeologs are highly conserved, and 10 haplotypes were revealed based on 19 single nucleotide polymorphisms (eight induced single amino acid residue substitutions, including 2 altered amino acids in transmembrane domains) in 80 widely distributed Ae. tauschii accessions. The results provide genetic resources for low/no Cd concentration wheat improvement.

Unique properties of Coronaviridae single-pass transmembrane domain regions as an adaptation to diverse membrane systems

Enveloped viruses such as Coronaviridae (CoV) enter the host cell by fusing the viral envelope directly with the plasma membrane (PM) or with the membrane of the endosome. Replication of the CoV genome takes place in membrane compartments formed by rearrangement of the endoplasmic reticulum (ER) membrane network. Budding of these viruses occurs from the ER-Golgi intermediate compartment (ERGIC). The relationship between proteins and various membranes is crucial for the replication cycle of CoVs. The role of transmembrane domains (TMDs) and pre-transmembrane domains (pre-TMD) of viral proteins in this process is gaining more recognition. Here we present a thorough analysis of physico-chemical parameters, such as accessible surface area (ASA), average hydrophobicity (Hav), and contribution of specific amino acids in TMDs and pre-TMDs of single-span membrane proteins of human viruses. We focus on unique properties of these elements in CoV and postulate their role in adaptation to diverse host membranes and regulation of retention of membrane proteins during replication.

The vacuolar membrane sucrose transporter MdSWEET16 plays essential roles in the cold tolerance of apple

Sugar content and cold tolerance are important apple (Malus × domestica) characteristics targeted by breeding programs. Here, a vacuolar membrane sucrose transporter, MdSWEET16, was cloned and characterized. A phylogenetic tree analysis found that MdSWEET16 was on the same polygenetic branch as Arabidopsis thaliana SWEET16 and SWEET17. MdSWEET16 was located on chromosome 2 and consists of six exons and five introns. The recombinant protein was obtained by prokaryotic induction, and the amino acid sequence and transmembrane domain were analyzed by bioinformatics. The encoded ~ 30-kDa protein has seven transmembrane domains and is localized on the tonoplasts of ‘Orin’ callus protoplasts. The expression of MdSWEET16 changed in response to sucrose and low temperature in ‘Orin’ calli. In addition, we also analyzed the expression level of MdSWEET16 during different fruit developmental stages using qRT-PCR. MdSWEET16 was highly expressed in young fruit, and its expression level during fruit development was significantly negatively correlated with sucrose content as assessed by a quantitative fluorescence analysis. Overexpression of MdSWEET16 in ‘Orin’ calli could reduce their sucrose content but increased their cold tolerance compared with MdSWEET16 RNA interference calli, which indicated that MdSWEET16 is involved in the sucrose transport and cold tolerance of apple. MdSWEET16’s expression level was significantly correlated with the sucrose content and low temperature, and was induced by sucrose and low temperatures. Overexpression of MdSWEET16 increased ‘Orin’ calli cold tolerance. The prokaryotic expression of its recombinant protein can be used to further study the function of the MdSWEET16 protein in apple.

A DedA Family Membrane Protein Is Required for Burkholderia thailandensis Colistin Resistance.

Colistin is a “last resort” antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ΔdbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ΔdbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.

In Silico Analysis of Single Nucleotide Polymorphisms (SNPs) in Human <i>MPL</i> Gene

Thrombopoietin was shown to be the major regulator of megakaryocytopoiesis and platelet formation. The protein encoded by the c-mpl gene, CD110, is a 635 amino acid transmembrane domain, with two extracellular cytokine receptor domains and two intracellular cytokine receptor box motifs. Mutations to this gene are associated with myelofibrosis and essential Thrombocythemia. In essential Thrombocythemia, these mutations lead to the production of Thrombopoietin receptors that are constitutively activated, or constantly turned on, which results in the overproduction of abnormal megakaryocytes. MPL gene was investigated in NCBI database (http://www.ncbi.nlm.nih.gov/) and computational software’s analyzed SNPs. SNPs in the coding region (exonal SNPs) that are non-synonymous (nsSNP) were analyzed by (sift, polyphen, Imutant and PHD-snp) softwares, and then SNPs at un-traslated region at 5’ ends (5UTR) were analyzed too by SNPs Function prediction software. In this study, Bioinformatics’ analysis of MPL gene initiated by SIFTand Polyphen-2server issued to review 197 SNPs and among this SNPs 23 pathological polymorphisms. Among these 23, 20 pathological polymorphisms were found to be very damaging, with higher Polyphen-2score, of the Polyphen-2 server (=1) and SIFT tolerance index of 0.000-0.005. Protein structural analysis was done by modeling of amino acid substitutions using Project Hope. AlsoI-Mutant software used to check their stability and the effect of the native and mutant residues protein and structure for all these pathological polymorphisms. We hope our results will provide useful information that is needed to help researchers to do further studies.

CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.

沙田柚钙调素结合蛋白(CaMBP)基因的鉴定与序列分析

利用高通量测序对沙田柚自交和异交花柱进行转录组测序，通过差异分析得到了沙田柚钙调素结合蛋白(CaMBP)基因序列。采用生物信息学方法，对其编码的蛋白质从序列特征、理化性质、跨膜结构域、高级结构以及功能域等方面进行了预测和分析。结果表明该基因全长为1616 bp，开放阅读框(ORF)全长为1368 bp，编码455个氨基酸，编码蛋白质的分子量为50.11 KDa，理论等电点为5.33。该蛋白质含有一个与钙调素结合蛋白相同的保守结构域。氨基酸序列比对结果显示，其编码的蛋白质与克莱门柚(Citrus clementina)的钙调结合蛋白相似性较高，相似度约为98%。这些分析结果可为今后深入研究该蛋白的结构特征和功能提供参考。 In this paper, the transcriptome of the self-pollinated style and cross-pollinated style of Citrus grandis var. Shatinyu Hort were sequenced by high-throughput sequencing technology. A calmo-dulin-binding protein gene sequence of Citrus grandis var. Shatinyu Hort was obtained though differential analysis method, and some characters of the calmodulin-binding protein were analyzed and predicted by bioinformatics method, including the composition of amino acid sequence, physicochemical parameters, hydrophobicity, transmembrane domain, secondary structure and function of protein etc. The results showed that the calmodulin-binding protein gene was 1616 bp in length with an open reading frame (ORF) of 1368 bp, encoding 455 amino acids with deduced molecular weight of 50.11 KDa, and theoretical pI value of 5.33, and contained a calmodulin-binding proteins domain. Bioinformatics analysis showed that the calmodulin-binding protein was a hydrophobic and unstable protein. The homology analysis of amino acid sequence indicated that the calmodulin-binding protein shared high homology with calmodulin-binding protein of Citrus clementina (98%). This work provides a systemic sequence analysis of the calmodulin-binding protein of Citrus grandis var. Shatinyu Hort. It will provide the useful reference for further investigation of its structure and function．

Cysteine substitution of transmembrane domain amino acids alters the ethanol inhibition of GluN1/GluN2A N-methyl-D-aspartate receptors.

N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors.

Molecular evolution of growth hormone and insulin-like growth factor 1 receptors in long-lived, small-bodied mammals

Mammals typically display a robust positive relationship between lifespan and body size. Two groups that deviate markedly from this pattern are bats and African mole-rats, with members of both groups being extremely long-lived given their body size, with the maximum documented lifespan for many species exceeding 20years. A recent genomics study of the exceptionally long-lived Brandt's bat, Myotis brandtii (41years), suggested that its longevity and small body size may be at least partly attributed to key amino acid substitutions in the transmembrane domains of the receptors of growth hormone (GH) and insulin-like growth factor 1 (IGF1). However, whereas elevated longevity is likely to be common across all 19 bat families, the reported amino acid substitutions were only observed in two closely related bat families. To test the hypothesis that an altered GH/IGF1 axis relates to the longevity of African mole-rats and bats, we compared and analysed the homologous coding gene sequences in genomic and transcriptomic data from 26 bat species, five mole-rats and 38 outgroup species. Phylogenetic analyses of both genes recovered the majority of nodes in the currently accepted species tree with high support. Compared to other clades, such as primates and carnivores, the bats and rodents had longer branch lengths. The single 24 amino acid transmembrane domain of IGF1R was found to be more conserved across mammals compared to that of GHR. Within bats, considerable variation in the transmembrane domain of GHR was found, including a previously unreported deletion in Emballonuridae. The transmembrane domains of rodents were found to be more conserved, with mole-rats lacking uniquely conserved amino acid substitutions. Molecular evolutionary analyses showed that both genes were under purifying selection in bats and mole-rats. Our findings suggest that while the previously documented mutations may confer some additional lifespan to Myotis bats, other, as yet unknown, genetic differences are likely to account for the long lifespans observed in many bat and mole-rat species.

Proline localized to the interaction interface can mediate self-association of transmembrane domains

Assembly of transmembrane domains (TMDs) is a critical step in the function of membrane proteins. In recent years, the role of specific amino acids in TMD–TMD interactions has been better characterized, with more emphasis on polar and aromatic residues. Despite the high abundance of proline residues in TMDs, contribution of proline to TMD–TMD association has not been intensively studied. Here, we evaluated statistically the frequency of appearance, and experimentally the contribution of proline, compared to other hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, and Met), with regard to TMD–TMD self-assembly. Our model system is the assembly motif (22QxxS25) found previously in TMDs of the Escherichia coli aspartate receptor (Tar-1). Statistically, our data revealed that all different motifs, except PxxS (P/S), have frequencies similar to their theoretical random expectancy within a database of 41916 sequences of TMDs, while PxxS motif is underrepresented. Experimentally, using the ToxR assembly system, the SDS-gel running pattern of biotin-conjugated TMD peptides, and FRET experiments between fluorescence-labeled peptides, we found that only the P/S motif preserves the dimerization ability of wild-type Tar-1 TMD. Although proline is known as a helix breaker in solution, Circular Dichroism spectroscopy revealed that the secondary structure of the P/S and the wild-type peptides are similar. All together, these data suggest that proline can stabilize TM self-assembly when localized to the interaction interface of a transmembrane oligomer. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Cysteine Scanning Mutagenesis on Transmembrane Domain 1 of the Liver‐Specific Organic Anion Transporting Polypeptide 1B3

The liver-specific organic anion transporting polypeptide 1B3 (OATP1B3) is a multispecific transporter that mediates the uptake of a broad range of drugs and other xenobiotics into hepatocytes. To identify regions in OATP1B3 important for substrate binding and/or translocation, we performed cysteine scanning mutagenesis. Twenty-two amino acids in transmembrane domain 1 (TM1) and extracellular loop 1 (EL1) were individually mutated to cysteines, and mutant proteins were transiently expressed in HEK293 cells. To determine the effect of each mutation on protein function, we measured uptake of estrone-3-sulfate and estradiol-17β-glucuronide. Transport was significantly reduced in several mutants after correcting for surface expression, indicating that this stretch of amino acids may be important for protein function. Functional cysteine mutants were then incubated with the cysteine-modifying reagents MTSES or MTSET prior to measuring uptake. MTS pre-incubation strongly inhibited the function of those mutants which could be labeled with biotinylated maleimide. Preincubating cells with estradiol-17β-glucuronide prior to labeling with biotinylated maleimide reduced the extent to which several mutants were labeled. These data suggest that TM1 and EL1 in OATP1B3 play a significant role in substrate binding and/or translocation. Supported by NIH grants RR021940, GM077336.

The Transmembrane Domain of the Molecular Chaperone Cosmc Directs Its Localization to the Endoplasmic Reticulum

The molecular basis for retention of integral membrane proteins in the endoplasmic reticulum (ER) is not well understood. We recently discovered a novel ER molecular chaperone termed Cosmc, which is essential for folding and normal activity of the Golgi enzyme T-synthase. Cosmc, a type II single-pass transmembrane protein, lacks any known ER retrieval/retention motifs. To explore specific ER localization determinants in Cosmc we generated a series of Cosmc mutants along with chimeras of Cosmc with a non-ER resident type II protein, the human transferrin receptor. Here we show that the 18 amino acid transmembrane domain (TMD) of Cosmc is essential for ER localization and confers ER retention to select chimeras. Moreover, mutations of a single Cys residue within the TMD of Cosmc prevent formation of disulfide-bonded dimers of Cosmc and eliminate ER retention. These studies reveal that Cosmc has a unique ER-retention motif within its TMD and provide new insights into the molecular mechanisms by which TMDs of resident ER proteins contribute to ER localization.

Identification of Sterol-Sensing Amino Acids in Transmembrane Domain 2 of the BK Channel Beta 1 subunit

The activity of large conductance, voltage- and calcium-gated potassium (BK) channels in myocytes critically controls arterial diameter. At <300 μM in the aqueous phase (below critical micellar concentration), lithocholate (LC) and related steroids increase myocyte BK channel activity (Po) and cause arterial dilation. We showed that: 1) BK β1 TM2 is critical for the channel sensitivity to LC, and 2) BK β2-4 cannot substitute for β1 in providing LC-sensitivity to BK channels (Bukiya et al., 2008; 2009). Moreover, computational dynamics identified two LC-sensing amino acid clusters in BK β1TM2: L157,L158,T165 and T169,L172,L173. Here, we used pinpoint mutagenesis and patch-clamp electrophysiology to determine the relative contribution of each cluster to the LC-sensitivity of BK channels. BK α (cbv1) and β1 subunits cloned from rat cerebral artery myocytes (AY330293; FJ154955) were coexpressed in Xenopus oocytes, and BK currents were recorded from inside-out patches at Ca2+i=10 μM and Vm=-20 to −40mV. BK β1T165A and β1T165A,L157A,L158A mutants rendered LC-sensitive BK channels, with 150 μM LC-induced increase in Po (×1.7 times) being identical to that found with cbv1+wtβ1. In contrast, BK β1T165A,T169A and β1T169A mutants were absolutely unresponsive to ≥150 μM LC. Data unveil a critical role for β1T169 in providing LC-sensitivity to BK channels. While the β1T169S mutation was able to rescue the channel's LC-sensitivity, the β1T169S,L172A,L173A mutation failed to do so. Moreover, BK β1L172A,L173A was also LC-insensitive. These data validate our computational prediction that hydrogen bonding between T/S169 and the steroid hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for successful docking of LC onto the T169,L172,L173 cluster. Thus, T169, L172 and L173 in BK β1TM2 provide a docking surface that is essential for the LC-sensitivity of BK channels.Support: HL104631 (AMD).

Comparison of the pharmacological properties of human and rat histamine H 3-receptors

Ligand pharmacology of histamine H 3-receptors is species-dependent. In previous studies, two amino acids in transmembrane domain 3 (TM III) were shown to play a significant role. In this study, we characterized human and rat histamine H 3-receptors (hH 3R and rH 3R, respectively), co-expressed with mammalian G proteins in Sf9 insect cell membranes. We compared a series of imidazole-containing H 3R ligands in radioligand binding and steady-state GTPase assays. H 3Rs similarly coupled to Gα i/o-proteins. Affinities and potencies of the agonists histamine, N α-methylhistamine and R-(α)-methylhistamine were in the same range. Imetit was only a partial agonist. The pharmacology of imetit and proxifan was similar at both species. However, impentamine was more potent and efficacious at rH 3R. The inverse agonists ciproxifan and thioperamide showed higher potency but lower efficacy at rH 3R. Clobenpropit was not species-selective. Strikingly, imoproxifan was almost full agonist at hH 3R, but an inverse agonist at rH 3R. Imoproxifan was docked into the binding pocket of inactive and active hH 3R- and rH 3R-models and molecular dynamic simulations were performed. Imoproxifan bound to hH 3R and rH 3R in E-configuration, which represents the trans-isomer of the oxime-moiety as determined in crystallization studies, and stabilized active hH 3R-, but inactive rH 3R-conformations. Large differences in electrostatic surfaces between TM III and TM V cause differential orientation of the oxime-moiety of imoproxifan, which then differently interacts with the rotamer toggle switch Trp 6.48 in TM VI. Collectively, the substantial species differences at H 3Rs are explained at a molecular level by the use of novel H 3R active-state models.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates

The human reduced folate carrier (hRFC) facilitates membrane transport of folates and antifolates. hRFC is characterized by 12 transmembrane domains (TMDs). To identify residues or domains involved in folate binding, we used substituted cysteine (Cys) accessibility methods (SCAM) with sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES). We previously showed that residues in TMD11 of hRFC were involved in substrate binding, whereas those in TMD12 were not (Hou, Z., Stapels, S. E., Haska, C. L., and Matherly, L. H. (2005) J. Biol. Chem. 280, 36206-36213). In this study, 232 Cys-substituted mutants spanning TMDs 1-10 and conserved stretches within the TMD6-7 (residues 204-217) and TMD10-11 connecting loop domains were transiently expressed in hRFC-null HeLa cells. All Cys-substituted mutants showed moderate to high levels of expression on Western blots, and only nine mutants including R133C, I134C, A135C, Y136C, S138C, G163C, Y281C, R373C, and S313C were inactive for methotrexate transport. MTSES did not inhibit transport by any of the mutants in TMDs 1, 3, 6, and 9 or for positions 204-217. Whereas most of the mutants in TMDs 2, 4, 5, 7, 8, and 10, and in the TMD10-11 connecting loop were insensitive to MTSES, this reagent inhibited methotrexate transport (25-75%) by 26 mutants in these TMDs. For 13 of these (Y126C, S137C, V160C, S168C, W274C, S278C, V284C, V288C, A311C, T314C, Y376C, Q377C, and V380C), inhibition was prevented by leucovorin, another hRFC substrate. Combined with our previous findings, these results implicate amino acids in TMDs 4, 5, 7, 8, 10, and 11, but not in TMDs 1, 2, 3, 6, 9, or 12, as important structural or functional components of the putative hydrophilic cavity for binding of anionic folate substrates.

Fast and Quantitative Recovery of Hydrophobic and Amphipathic Peptides after Incorporation into Phospholipid Membranes

A new method that allows fast and quantitative recovery of hydrophobic or amphipathic peptides, or both, after their intimate incorporation into lipid membranes, is proposed. It relies on the use of small Sep-Pak cartridges and simple chromatographic handling. Peptides selected for this study are the 35 amino acid transmembrane domain of the Neu/erbB-2 protein and its point mutated (V664E) analogue expressed in some cancers, the 25 amino acid BH4 domain from the Bcl-2 antiapoptotic protein and the 15 amino acid Catestatin segment from chromogranin A found to have antimicrobial capabilities. Incorporation of peptides into membranes is accomplished using organic solvent cosolubilization and several cycles of freeze-drying/hydration from aqueous solution. For the hydrophobic peptides, separation from the membrane is performed on Sep-Pak C2 columns in two steps: (i) water/methanol elution of lipids and (ii) peptide elution using aprotic solvents (acetonitrile, 2-propanol). For amphipathic peptides, separation is performed on Sep-Pak C(18) columns using selective elution in one single step: water/methanol elution to recover first the peptide and then the lipids. Peptide and lipid recovery after all purification steps range from 60 to 80%, with peptide purity above 96%. This new method is simple, inexpensive, and very fast: a 10-mg membranous mixture containing 10% (w/w) peptide may be separated in 20-30 min.

Molecular Characterization of Human Melanocortin-3 Receptor Ligand−Receptor Interaction

Melanocortin-3 receptor (MC3R), primarily expressed in the hypothalamus, plays an important role in the regulation of energy homeostasis. MC3R-deficient (MC3R(-)(/)(-)) mice demonstrate increased fat mass, higher feeding efficiency, hyperleptinaemia, and mild hyperinsulinism. At least one specific mutation of MC3R has been identified to be associated with human obesity. Functional analysis of this altered MC3R (I183N) has indicated that the mutation completely abolishes agonist-mediated receptor activation. However, the specific molecular determinants of MC3R responsible for ligand binding and receptor signaling are currently unknown. The present study is to determine the structural aspects of MC3R responsible for ligand binding and receptor signaling. On the basis of our theoretical model for MC1R, using mutagenesis, we have examined 19 transmembrane domain amino acids selected for these potential roles in ligand binding and receptor signaling. Our results indicate that (i) substitutions of charged amino acid residues E131 in transmembrane domain 2 (TM2), D154 and D158 in TM3, and H298 in TM6 with alanine dramatically reduced NDP-MSH binding affinity and receptor signaling, (ii) substitutions of aromatic amino acids F295 and F296 in TM6 with alanine also significantly decreased NDP-MSH binding and receptor activity, (iii) substitutions of D121in TM2 and D332 in TM7 with alanine resulted in the complete loss of ligand binding, ligand induced receptor activation, and cell surface protein expression, and (iv) interestingly, substitution of L165 in TM3 with methionine or alanine switched antagonist SHU9119 into a receptor agonist. Our results suggest that TM3 and TM6 are important for NDP-MSH binding, while D121 in TM2 and D332 in TM7 are crucial for receptor activity and signaling. Importantly, L165 in TM3 is critical for agonist or antagonist selectivity. These results provide important information about the molecular determinants of hMC3R responsible for ligand binding and receptor signaling.

Molecular Characterization of the Gerbil C5a Receptor and Identification of a Transmembrane Domain V Amino Acid That Is Crucial for Small Molecule Antagonist Interaction

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.