RESUMO
The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.
Assuntos
Adesinas de Escherichia coli , Receptor de Manose , Modelos Moleculares , Humanos , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Manose/metabolismo , Receptor de Manose/química , Receptor de Manose/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Simulação de Acoplamento MolecularRESUMO
Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcusâ pneumoniae, Vibrioâ cholerae, or Clostridiumâ perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S.â pneumoniae (NanA) and the symbiotic microorganism B.â thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.
Assuntos
Neuraminidase/antagonistas & inibidores , Streptococcus pneumoniae/enzimologia , Domínio Catalítico/efeitos dos fármacos , Neuraminidase/metabolismo , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Human norovirus binding to histo-blood group antigens (HBGAs) is thought to direct their entry into host cells. However, the glycan epitopes characteristic of HBGAs are also present on oligosaccharides abundant in human milk. In this issue of JBC, Hanisch et al compared norovirus binding to human gastric mucins and human milk oligosaccharides, finding those bound most avidly are rich in α-fucose. Mimicry of these epitopes with α-fucose multivalently displayed on other carbohydrate scaffolds successfully scavenged this prevalent virus, providing new insights into norovirus biology and clues for future therapeutic development.
Assuntos
Infecções por Caliciviridae/imunologia , Fucose/imunologia , Leite Humano/imunologia , Norovirus/imunologia , Oligossacarídeos/imunologia , Sítios de Ligação , Epitopos/química , Epitopos/imunologia , Fucose/análogos & derivados , Humanos , Leite Humano/química , Mucinas/química , Mucinas/imunologia , Norovirus/fisiologia , Oligossacarídeos/química , Polissacarídeos/química , Polissacarídeos/imunologia , Internalização do VírusRESUMO
Bacterial pathogens are a major cause of foodborne diseases and food poisoning. To cope with the acid conditions encountered in different environments such as in fermented food or in the gastric compartment, neutralophilic bacteria have developed several adaptive mechanisms. One of those mechanisms, the amino acid dependent system, consumes intracellular protons in biochemical reactions. It involves an antiporter that facilitates the exchange of external substrate amino acid for internal product and a cytoplasmic decarboxylase that catalyzes a proton-consuming decarboxylation of the substrate. So far, four acid resistance antiporters have been discovered, namely the glutamate-γ-aminobutyric acid antiporter GadC, the arginine-agmatine antiporter AdiC, the lysine-cadaverine antiporter CadB, and the ornithine-putrescine antiporter PotE. The 3D structures of AdiC and GadC, reveal an inverted-repeat fold of two times 5 transmembrane helices, typical of the amino acid-polyamine-organocation (APC) superfamily of transporters. This review summarizes our current knowledge on the transport mechanism, the pH regulation and the selectivity of these four acid resistance antiporters. It also highlights that AdiC is a paradigm for eukaryotic amino acid transporters of the APC superfamily as structural models of several of these transporters built using AdiC structures were exploited to unveil their mechanisms of amino acid recognition and translocation.
Assuntos
Antiporters/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer's disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.
Assuntos
Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Apolipoproteína E4/fisiologia , Apolipoproteínas E/química , Humanos , Ligantes , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Espectrometria de Massas , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/químicaRESUMO
Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.
Assuntos
Adesinas de Escherichia coli/química , Antibacterianos/farmacologia , Escherichia coli/patogenicidade , Proteínas de Fímbrias/química , Antibacterianos/química , Aderência Bacteriana/efeitos dos fármacos , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Fímbrias/antagonistas & inibidores , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
The fimbrial lectin FimH from uro- and enteropathogenic Escherichia coli binds with nanomolar affinity to oligomannose glycans exposing Manα1,3Man dimannosides at their non-reducing end, but only with micromolar affinities to Manα1,2Man dimannosides. These two dimannoses play a significantly distinct role in infection by E. coli. Manα1,2Man has been described early on as shielding the (Manα1,3Man) glycan that is more relevant to strong bacterial adhesion and invasion. We quantified the binding of the two dimannoses (Manα1,2Man and Manα1,3Man to FimH using ELLSA and isothermal microcalorimetry and calculated probabilities of binding modes using molecular dynamics simulations. Our experimentally and computationally determined binding energies confirm a higher affinity of FimH towards the dimannose Manα1,3Man. Manα1,2Man displays a much lower binding enthalpy combined with a high entropic gain. Most remarkably, our molecular dynamics simulations indicate that Manα1,2Man cannot easily take its major conformer from water into the FimH binding site and that FimH is interacting with two very different conformers of Manα1,2Man that occupy 42% and 28% respectively of conformational space. The finding that Manα1,2Man binding to FimH is unstable agrees with the earlier suggestion that E. coli may use the Manα1,2Man epitope for transient tethering along cell surfaces in order to enhance dispersion of the infection.
Assuntos
Adesinas de Escherichia coli/química , Proteínas de Fímbrias/química , Lectinas/química , Polissacarídeos/química , Aderência Bacteriana , Calorimetria , Escherichia coli/fisiologia , Lectinas/metabolismo , Manose/química , Modelos Moleculares , Conformação Molecular , Ligação Proteica , TermodinâmicaRESUMO
The voltage-dependent anion-selective channel (VDAC) is the main pathway for inorganic ions and metabolites through the mitochondrial outer membrane. Studies recently demonstrated that membrane lipids regulate its function. It remains, however, unclear how this regulation takes place. In this study, we show that phospholipids are key regulators of Phaseolus VDAC function and, furthermore, that the salt concentration modulates this regulation. Both selectivity and voltage dependence of Phaseolus VDAC are very sensitive to a change in the lipid polar head from PC to PE. Interestingly enough, this dependence is observed only at low salt concentration. Furthermore, significant changes in VDAC functional properties also occur with the gradual methylation of the PE group pointing to the role of subtle chemical variations in the lipid head group. The dependence of PcVDAC gating upon the introduction of a small mole fraction of PE in a PC bilayer has prompted us to propose the existence of a specific interaction site for PE on the outer surface of PcVDAC. Eventually, comparative modeling and molecular dynamics simulations suggest a potential mechanism to get insight into the anion selectivity enhancement of PcVDAC observed in PE relative to PC.
Assuntos
Transporte de Íons , Lipídeos de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Phaseolus/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Plantas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Sementes/metabolismo , Relação Estrutura-AtividadeRESUMO
Antagonists of the Escherichia coli type-1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against acute and recurrent bacterial infections. In this study α-d-mannopyranosides O- or C-linked with an alkyl, alkene, alkyne, thioalkyl, amide, or sulfonamide were investigated to fit a hydrophobic substituent with up to two aryl groups within the tyrosine gate emerging from the mannose-binding pocket of FimH. The results were summarized into a set of structure-activity relationships to be used in FimH-targeted inhibitor design: alkene linkers gave an improved affinity and inhibitory potential, because of their relative flexibility combined with a favourable interaction with isoleucine-52 located in the middle of the tyrosine gate. Of particular interest is a C-linked mannoside, alkene-linked to an ortho-substituted biphenyl that has an affinity similar to its O-mannosidic analog but superior to its para-substituted analog. Docking of its high-resolution NMR solution structure to the FimH adhesin indicated that its ultimate, ortho-placed phenyl ring is able to interact with isoleucine-13, located in the clamp loop that undergoes conformational changes under shear force exerted on the bacteria. Molecular dynamics simulations confirmed that a subpopulation of the C-mannoside conformers is able to interact in this secondary binding site of FimH.
Assuntos
Adesinas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Manosídeos/farmacologia , Adesinas de Escherichia coli/química , Aderência Bacteriana , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Proteínas de Fímbrias/química , Manosídeos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E.â coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10â mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Doença de Crohn/terapia , Escherichia coli/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Manosídeos/farmacologia , Adesinas de Escherichia coli/metabolismo , Animais , Disponibilidade Biológica , Peso Corporal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteínas de Fímbrias/antagonistas & inibidores , Proteínas de Fímbrias/metabolismo , Humanos , Manosídeos/química , Manosídeos/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Domínios ProteicosRESUMO
Amino acids constitute a major nutritional source for probably all fungi. Studies of model species such as the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans have shown that they possess multiple amino acid transporters. These proteins belong to a limited number of superfamilies, now defined according to protein fold in addition to sequence criteria, and differ in subcellular location, substrate specificity range, and regulation. Structural models of several of these transporters have recently been built, and the detailed molecular mechanisms of amino acid recognition and translocation are now being unveiled. Furthermore, the particular conformations adopted by some of these transporters in response to amino acid binding appear crucial to promoting their ubiquitin-dependent endocytosis and/or to triggering signaling responses. We here summarize current knowledge, derived mainly from studies on S. cerevisiae and A. nidulans, about the transport activities, regulation, and sensing role of fungal amino acid transporters, in relation to predicted structure.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Aspergillus nidulans/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/química , Aspergillus nidulans/genética , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H(+)-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H(+) coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Arginina/metabolismo , Sítios de Ligação/genética , Transporte Biológico , Imageamento Tridimensional , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
The voltage-dependent anion channel (VDAC) serves as the major pore for metabolites and electrolytes in the outer mitochondrial membrane. To refine our understanding of ion permeation through this channel we performed an extensive Brownian (BD) and molecular dynamics (MD) study on the mouse VDAC isoform 1 wild-type and mutants (K20E, D30K, K61E, E158K and K252E). The selectivity and the conductance of the wild-type and of the variant channels computed from the BD trajectories are in agreement with experimental data. The calculated selectivity is shown to be very sensitive to slight conformational changes which may have some bearing on the variability of the selectivity values measured on the VDAC open state. The MD and BD free energy profiles of the ion permeation suggest that the pore region comprising the N-terminal helix and the barrel band encircling it predominantly controls the ion transport across the channel. The overall 12µs BD and 0.9µs MD trajectories of the mouse VDAC isoform 1 wild-type and mutants feature no distinct pathways for ion diffusion and no long-lived ion-protein interactions. The dependence of ion distribution in the wild-type channel with the salt concentration can be explained by an ionic screening of the permanent charges of the protein arising from the pore. Altogether these results bolster the role of electrostatic features of the pore as the main determinant of VDAC selectivity towards inorganic anions.
Assuntos
Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/fisiologia , Animais , Condutividade Elétrica , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Cloreto de Potássio/farmacologia , Estrutura Terciária de Proteína , Eletricidade Estática , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/fisiologiaRESUMO
The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Isoenzimas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Nucleotídeos de Adenina/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Guanina/metabolismo , Humanos , Lactococcus lactis/metabolismo , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Transporte Proteico , Especificidade por SubstratoRESUMO
The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and the major transport pathway for a large variety of compounds ranging from ions to large polymeric molecules such as DNA and tRNA. Plant VDACs feature a secondary structure content and electrophysiological properties akin to those of VDACs from other organisms. They however undergo a specific regulation. The general importance of VDAC in plant physiology has only recently emerged. Besides their role in metabolite transport, plant VDACs are also involved in the programmed cell death triggered in response to biotic and abiotic stresses. Moreover, their colocalization in non-mitochondrial membranes suggests a diversity of function. This review summarizes our current understanding of the structure and function of plant VDACs. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
Assuntos
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Sequência de Aminoácidos , DNA de Plantas/metabolismo , Fenômenos Eletrofisiológicos , Dados de Sequência Molecular , Proteínas de Plantas/genética , RNA de Plantas/metabolismo , Canais de Ânion Dependentes de Voltagem/químicaRESUMO
The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.
Assuntos
Nucleotídeos de Adenina/química , Cloretos/química , Translocases Mitocondriais de ADP e ATP/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis , Proteínas de Arabidopsis/química , Bovinos , Simulação por Computador , Sequência Conservada , Campos Eletromagnéticos , Escherichia coli , Translocases Mitocondriais de ADP e ATP/genética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Eletricidade Estática , Fatores de TempoRESUMO
The bacterial photosynthetic reaction center is the evolutionary ancestor of the Photosystem II reaction center. These proteins share the same fold and perform the same biological function. Nevertheless, the details of their molecular reaction mechanism differ. It is of significant biological and biochemical interest to determine which functional characteristics are conserved at the level of the protein sequences. Since the level of sequence identity between the bacterial photosynthetic reaction center and Photosystem II is low, a progressive multiple-sequence alignment leads to errors in identifying the conserved residues. In such a situation, profile hidden Markov models (pHMM) can be used to obtain reliable multiple-sequence alignments. We therefore constructed the pHMM with the help of a sequence alignment based on a structural superposition of both proteins. To validate the multiple-sequence alignments obtained with the pHMM, the conservation of residues with known functional importance was examined. Having confirmed the correctness of the multiple-sequence alignments, we analyzed the conservation of residues involved in hydrogen bonding and redox potential tuning of the cofactors. Our analysis reveals similarities and dissimilarities between the bacterial photosynthetic reaction center and Photosystem II at the protein sequence level, hinting at different charge separation and charge transfer mechanisms. The conservation analysis that we perform in this paper can be considered as a model for analyzing the conservation in proteins with a low level of sequence identity.
Assuntos
Cadeias de Markov , Modelos Moleculares , Complexo de Proteína do Fotossistema II/química , Rhodobacter sphaeroides/química , Sequência de Aminoácidos , Bacterioclorofilas/química , Sítios de Ligação , Coenzimas , Sequência Conservada , Transporte de Elétrons , Ligação de Hidrogênio , Dados de Sequência Molecular , Pigmentos Biológicos/química , Subunidades Proteicas/química , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Determination of glycosidase hydrolysis kinetics for a monovalent sugar substrate is relatively straightforward and classically achieved by monitoring the fluorescence signal released from the sugar-conjugated probe after enzymatic hydrolysis. Naturally occuring sugar epitopes are, however, often clustered on biopolymers or at biological surfaces, and previous reports have shown that glycosidase hydrolytic rates can differ greatly with multivalent presentation of the sugar epitopes. New probes are needed to make it easier to interpret the importance of substrate clustering towards a specific enzyme activity. In this work, we developed multivalent glucuronide substrates attached to fluorescent amino-coumarines through self-immolative linkers to enable real time-monitoring of the hydrolysing activity of E.coli ß-glucuronidases (GUS) towards clustered substrates. GUS are exoglycosidases of considerable therapeutic interest cleaving ß-d-glucuronides and are found in the lysosomes, in the tumoral microenvironment, and are expressed by gut microbiota. GUS showed a much lower catalytic efficiency in hydrolysing clustered glucuronides due to a significantly lower enzymatic velocity and affinity for the substrates. GUS was 52-fold less efficient in hydrolysing GlcA substrates presented on an octameric silsequioxane (COSS) compared with a monovalent GlcA of similar chemical structure. Thus, kinetic and thermodynamic data of GUS hydrolysis towards multivalent glucuronides were easily obtained with these new types of enzymatically-triggered probes. More generally, adapting the substrate nature and valency of these new probes, should improve understanding of the impact of multivalency for a specific enzyme.
RESUMO
The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 A, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (QB). We observe a new orientation of QB in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of QB in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxo-groups of QB in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of QB upon reduction.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Quinonas/química , Quinonas/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Metabolismo dos Lipídeos , Lipídeos/química , Modelos Moleculares , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Terciária de Proteína , Prótons , Rhodobacter sphaeroides/genética , Fatores de TempoRESUMO
The arginine-agmatine antiporter (AdiC) is a component of an acid resistance system developed by enteric bacteria to resist gastric acidity. In order to avoid neutral proton antiport, the monovalent form of arginine, about as abundant as its divalent form under acidic conditions, should be selectively bound by AdiC for transport into the cytosol. In this study, we shed light on the mechanism through which AdiC distinguishes Arg+ from Arg2+ of arginine by investigating the binding of both forms in addition to that of divalent agmatine, using a combination of molecular dynamics simulations with molecular and quantum mechanics calculations. We show that AdiC indeed preferentially binds Arg+. The weaker binding of divalent compounds results mostly from their greater tendency to remain hydrated than Arg+. Our data suggests that the binding of Arg+ promotes the deprotonation of Glu208, a gating residue, which in turn reinforces its interactions with AdiC, leading to longer residence times of Arg+ in the binding site. Although the total electric charge of the ligand appears to be the determinant factor in the discrimination process, two local interactions formed with Trp293, another gating residue of the binding site, also contribute to the selection mechanism: a cation-π interaction with the guanidinium group of Arg+ and an anion-π interaction involving Glu208.