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The kidney is a vital organ for the elimination of therapeutic drugs and their metabolites. Renal drug transporters, which are primarily located in the renal proximal tubules, play an important role in tubular secretion and reabsorption of drug molecules in the kidney. Tubular secretion is characterized by high clearance capacities, broad substrate specificities, and distinct charge selectivity for organic cations and anions. In the past two decades, substantial progress has been made in understanding the roles of transporters in drug disposition, efficacy, toxicity and drug–drug interactions (DDIs). In the kidney, several transporters are involved in renal handling of organic cation (OC) and organic anion (OA) drugs. These transporters are increasingly recognized as the target for clinically significant DDIs. This review focuses on the functional characteristics of major human renal drug transporters and their involvement in clinically significant DDIs., Graphical abstract Renal drug transporters, expressed in the basolateral and apical membrane of renal proximal tubules, play an important role in tubular secretion and reabsorption of drug molecules in the kidney. These transporters are increasingly recognized as the target for clinically significant drug–drug interactions.

Viral channel forming proteins (VCPs) have been discovered in the late 70s and are found in many viruses to date. Usually they are small and have to assemble to form channels which depolarize the lipid membrane of the host cells. Structural information is just about to emerge for just some of them. Thus, computational methods play a pivotal role in generating plausible structures which can be used in the drug development process. In this review the accumulation of structural data is introduced from a historical perspective. Computational performances and their predictive power are reported guided by biological questions such as the assembly, mechanism of function and drug–protein interaction of VCPs. An outlook of how coarse grained simulations can contribute to yet unexplored issues of these proteins is given. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov., Graphical abstract, Highlights • Early references about the discovery of viral channel forming proteins. • Latest structural information about the class of proteins. • Identification of structural motifs, assembly mechanism of function and drug action using computational methods. • Outlook for the use of coarse grained techniques to address assembly and integration into cellular processes.

The Envelope (E) protein in SARS Coronavirus (CoV) is a small structural protein, incorporated as part of the envelope. A major fraction of the protein has been known to be associated with the host membranes, particularly organelles related to intracellular trafficking, prompting CoV packaging and propagation. Studies have elucidated the central hydrophobic transmembrane domain of the E protein being responsible for much of the viroporin activity in favor of the virus. However, newer insights into the organizational principles at the membranous compartments within the host cells suggest further complexity of the system. The lesser hydrophobic Carboxylic-terminal of the protein harbors interesting amino acid sequences- suggesting at the prevalence of membrane-directed amyloidogenic properties that remains mostly elusive. These highly conserved segments indicate at several potential membrane-associated functional roles that can redefine our comprehensive understanding of the protein. This should prompt further studies in designing and characterizing of effective targeted therapeutic measures., Graphical abstract Unlabelled Image, Highlights • The SARS CoV Envelope protein is a small structural protein of the virus, responsible for viroporin like activity. • Membrane- E protein interaction provides an useful insight into gaining mechanistic insight into its viroporin functions. • The central hydrophobic transmembrane domain of E protein, known to affect ion-channel formation. • The C-terminal region of the protein show further potential host-membrane directed functional roles. • The highly conserved amyloidogenic amino acid stretches of the C-terminal suggest for its contribution to CoV propagation.

Growth hormone (GH) and structurally related cytokines regulate a great number of physiological and pathological processes. They do this by coupling their single transmembrane domain (TMD) receptors to cytoplasmic tyrosine kinases, either as homodimers or heterodimers. Recent studies have revealed that many of these receptors exist as constitutive dimers rather than being dimerized as a consequence of ligand binding, which has necessitated a new paradigm for describing their activation process. In the present study, we describe a model for activation of the tyrosine kinase Janus kinase 2 (JAK2) by the GH receptor homodimer based on biochemical data and molecular dynamics simulations. Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm. This movement slides the pseudokinase inhibitory domain of one JAK kinase away from the kinase domain of the other JAK within the receptor dimer–JAK complex, allowing the two kinase domains to interact and trans-activate. This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation. We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

Graphical abstract, Highlights • Flatworm and roundworm glutamate-gated chloride channels (GluCls) were compared. • Several glutamate analogues activated both GluCls in the millimolar range. • Quisqualate selectively activated the flatworm GluCl. • Propofol and thymol inhibited both GluCls in the micromolar range., Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

Highlights • A R504H mutation in human proprotein convertase PC7 is associated with increased HDL and reduced triglycerides. • Wild-type PC7 and its R504H mutant have identical cellular enzymatic activities. • In situ hybridization revealed co-localization of mouse ApoF and PC7 mRNAs in liver. • WT and PC7 KO mice do not exhibit changes in circulating levels of insulin or glucose. • WT and PC7 KO mice do not exhibit changes in circulating levels of HDL, TG and LDL., A genome-wide association study suggested that a R504H mutation in the proprotein convertase PC7 is associated with increased circulating levels of HDL and reduced triglycerides in black Africans. Our present results show that PC7 and PC7-R504H exhibit similar processing of transferrin receptor-1, proSortilin, and apolipoprotein-F. Plasma analyses revealed no change in the lipid profiles, insulin or glucose of wild type and PC7 KO mice. Thus, the R504H mutation does not modify the proteolytic activity of PC7. The mechanisms behind the implication of PC7 in the regulation of human HDL, triglycerides and in modifying the levels of atherogenic small dense LDL remain to be elucidated.

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics., Graphical abstract Ivermectin (red spheres) binding to Cel-Pgp-1 (blue ribbon: N-term, yellow ribbon: C-term).Image 1, Highlights • Cel-Pgp-1 cristal provides a valuable tool to study anthelmintic interactions. • Cel-Pgp-1 recognizes a broad panel of anthelmintics, including macrocyclic lactones. • Cel-Pgp-1 is predicted to transport MLs and contribute to anthelmintic resistance. • Sugars in avermectins increase the affinity to Cel-Pgp-1 compared with moxidectin. • These data provide a basis for modelling ABC homologs in parasitic nematodes.

To cleave DNA, the Type III RM (restriction–modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.

Membrane-spanning α-helices represent major sites of protein-protein interaction in membrane protein oligomerization and folding. As such, these interactions may be of exquisite specificity. Specificity often rests on a complex interplay of different types of residues forming the helix-helix interfaces via dense packing and different non-covalent forces, including van der Waal’s forces, hydrogen bonding, charge-charge interactions, and aromatic interactions. These interfaces often contain complex residue motifs where the contribution of constituent amino acids depends on the context of the surrounding sequence. Moreover, transmembrane helix-helix interactions are increasingly recognized as being dynamic and dependent on the functional state of a given protein.

Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley–White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure–function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful., Directed evolution of Cys (cysteines) in the multidrug transporter Pgp identified novel non-conservative substitutions, revealed important domain interactions and produced a CL protein that is more robust and active than previous CL Pgp built solely on conservative alanine substitutions.

Cholesterol synthesis occurs in the ER (endoplasmic reticulum), where most of the cholesterogenic machinery resides. As membrane-bound proteins, their topology is difficult to determine, and thus their structures are largely unknown. To help resolve this, we focused on the final enzyme in cholesterol synthesis, DHCR24 (3β-hydroxysterol Δ24-reductase). Prediction programmes and previous studies have shown conflicting results regarding which regions of DHCR24 are associated with the membrane, although there was general agreement that this was limited to only the N-terminal portion. Here, we present biochemical evidence that in fact the majority of the enzyme is associated with the ER membrane. This has important consequences for the many functions attributed to DHCR24. In particular, those that suggest DHCR24 alters its localization within the cell should be reassessed in light of this new information. Moreover, we propose that the expanding database of post-translational modifications will be a valuable resource for mapping the topology of membrane-associated proteins, such as DHCR24, that is, flagging cytosolic residues accessible to modifying enzymes such as kinases and ubiquitin ligases., We present new findings indicating that the final enzyme in cholesterol synthesis is strongly associated with the endoplasmic reticulum membrane throughout its length, contrary to previous studies that indicated only the beginning of the enzyme was associated with the membrane.

Highlights ► Computational modeling of PEDV OFR3 protein. ► Wild-type PEDV ORF3 protein functions as an ion channel and regulates virus production. ► The truncated protein encoded by an attenuated-type PEDV ORF3 shows less channel activity., Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6 and N6–N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.

The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E protein's pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li+ partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking., Graphical abstract, Highlights • HCoV-229E ORF4a protein is expressed in the infected Huh-7 cells and localized in the ERGIC. • ORF4a forms homo-oligomers and serves as ion channels in both Xenopus oocytes and yeast. • ORF4a protein is a non-selective channel for monovalent cations. • HCoV-229E ORF4a protein acts as viroporins to regulate the virus production.