RESUMO
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Modelos Moleculares , Conformação Proteica , Simportadores/química , Simportadores/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos , Sítios de Ligação , Mutação , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , Simportadores/genéticaRESUMO
The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Proteínas de Membrana Transportadoras/química , Conformação ProteicaRESUMO
Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.
Assuntos
Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Dimerização , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
The Mmr multidrug efflux pump recognizes and actively extrudes a broad range of antimicrobial agents, and promotes the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Mmr is controlled by the TetR-like transcriptional regulator Rv3066, whose open reading frame is located downstream of the mmr operon. To understand the structural basis of Rv3066 regulation, we have determined the crystal structures of Rv3066, both in the absence and presence of bound ethidium, revealing an asymmetric homodimeric two-domain molecule with an entirely helical architecture. The structures underscore the flexibility and plasticity of the regulator essential for multidrug recognition. Comparison of the apo-Rv3066 and Rv3066-ethidium crystal structures suggests that the conformational changes leading to drug-mediated derepression is primarily due to a rigid body rotational motion within the dimer interface of the regulator. The Rv3066 regulator creates a multidrug-binding pocket, which contains five aromatic residues. The bound ethidium is found buried within the multidrug-binding site, where extensive aromatic stacking interactions seemingly govern the binding. In vitro studies reveal that the dimeric Rv3066 regulator binds to a 14-bp palindromic inverted repeat sequence in the nanomolar range. These findings provide new insight into the mechanisms of ligand binding and Rv3066 regulation.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis , Proteínas Repressoras/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cromatografia em Gel , Pegada de DNA , Proteínas de Ligação a DNA/química , Farmacorresistência Bacteriana Múltipla , Ensaio de Desvio de Mobilidade Eletroforética , Etídio/química , Polarização de Fluorescência , Sequências Repetidas Invertidas , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Conformação Proteica , Proteínas Repressoras/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the recently determined structures of Mfsd2a, the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.
Assuntos
Ácidos Graxos Ômega-3 , Simportadores , Simportadores/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Barreira Hematoencefálica/metabolismo , Transporte Biológico , Ácidos Docosa-Hexaenoicos , Lisofosfatidilcolinas/químicaRESUMO
Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.
Assuntos
Neoplasias , Inibidores de Proteínas Quinases , Receptores de Fatores de Crescimento de Fibroblastos , Animais , Ratos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Transdução de Sinais , Relação Estrutura-Atividade , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacosRESUMO
mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/química , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Simulação de Dinâmica Molecular , Domínios Proteicos , Células Sf9 , Spodoptera , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Arginina/metabolismo , Proteínas de Peixe-Zebra/química , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Conformação Proteica , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.Multidrug efflux pumps significantly contribute for bacteria resistance to antibiotics. Here the authors present the structure of Campylobacter jejuni CmeB pump combined with functional FRET assays to propose a transport mechanism where each CmeB protomers is functionally independent from the trimer.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana Transportadoras/genética , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/química , Parede Celular/metabolismo , Cristalografia por Raios X , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Regiões Promotoras Genéticas , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance-nodulation-cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a ß-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a ß-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein-membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this ß-barrel, outer membrane channel.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação ProteicaRESUMO
As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance-nodulation-cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/metabolismo , Cristalografia por Raios X , Canais Iônicos/química , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Cisteína/metabolismo , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Fatores de Transcrição/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Modelos Moleculares , Neisseria gonorrhoeae/metabolismo , Conformação Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , HumanosRESUMO
Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Neisseria gonorrhoeae/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de SequênciaRESUMO
Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB(6)-CusA(3). We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.
Assuntos
Aminoácidos/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Aminoácidos/genética , Transporte Biológico/genética , Cristalografia por Raios X/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transporte de Íons , Proteínas de Fusão de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Mutação , Periplasma/genética , Periplasma/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli. We also determined the co-crystal structure of the CusBA adaptor-transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Sítios de Ligação , Membrana Celular/química , Cobre/química , Cristalografia por Raios X , Citoplasma/química , Escherichia coli/química , Metionina/química , Periplasma/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Prata/química , Relação Estrutura-AtividadeRESUMO
The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.