RESUMO
The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.
Assuntos
Inseticidas , Toxinas Biológicas , Bandagens , Transporte Biológico , Cristalografia por Raios X , Dobramento de ProteínaRESUMO
Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.
Assuntos
Apolipoproteína A-I , Colesterol , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Transporte Biológico , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fosfolipídeos/metabolismoRESUMO
Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Peixes/metabolismo , Receptores Purinérgicos P1/metabolismo , Glândula de Sal/metabolismo , Squalus acanthias/metabolismo , Animais , Clonagem Molecular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Evolução Molecular , Feminino , Proteínas de Peixes/genética , Humanos , Masculino , Potenciais da Membrana , Filogenia , Conformação Proteica , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/genética , Squalus acanthias/genética , Relação Estrutura-Atividade , Xenopus laevisRESUMO
The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11, derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.
RESUMO
Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes phosphodiester-linked adducts from both ends of DNA. This includes the topoisomerase I (TOP1)-DNA covalent reaction intermediate that is the target of the camptothecin class of chemotherapeutics. Tdp1 two-step catalysis is centered on the formation of a Tdp1-DNA covalent complex (Tdp1cc) using two catalytic histidines. Here, we examined the role of the understudied, structurally undefined, and poorly conserved N-terminal domain (NTD) of Tdp1 in context of full-length protein in its ability to remove TOP1cc in cells. Using toxic Tdp1 mutants, we observed that the NTD is critical for Tdp1's ability to remove TOP1-DNA adducts in yeast. Full-length and N-terminal truncated Tdp1 mutants showed similar expression levels and cellular distribution yet an inversed TOP1-dependent toxicity. Single turnover catalysis was significantly different between full-length and truncated catalytic mutants but not wild-type enzyme, suggesting that Tdp1 mutants depend on the NTD for catalysis. These observations suggest that the NTD plays a critical role in the regulation of Tdp1 activity and interaction with protein-DNA adducts such as TOP1cc in cells. We propose that the NTD is a regulatory domain and coordinates stabilization of the DNA-adducted end within the catalytic pocket to access the phosphodiester linkage for hydrolysis.
Assuntos
Adutos de DNA , DNA Topoisomerases Tipo I , Diester Fosfórico Hidrolases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.
Assuntos
Proteínas de Transporte de Cátions/química , Sítios de Ligação/genética , Proteínas de Transporte de Cátions/genética , Cobre/química , Transportador de Cobre 1 , Microscopia Crioeletrônica , Cristalização , Humanos , Mutação , Conformação ProteicaRESUMO
ABCA1 and ABCA4 are enigmatic because they transport substrates in opposite directions yet share >50% amino acid identity. ABCA4 imports lipid conjugates but ABCA1 exports lipids. Both hydrolyze ATP to drive transport, and both contain cytoplasmic regulatory domains (RDs) following nucleotide-binding domains (NBDs) in the primary structure. The tertiary structures of several ABC importers, including ABCA4, show that each RD forms a domain-swapped latch that locks onto the opposing RD and holds the NBDs close together. Crucially, sequences encoding the RDs and their bridges are among the most conserved in the entire ABC-A subfamily. In the original cryo-EM structure of ABCA1, the RDs were modeled without crossover. After close inspection of that cryo-EM density map and the recent structure of ABCA4, we propose that the RDs of ABCA1 also form a domain-swapped latch. A refined ABCA1 model containing latches exhibited significantly improved overall protein geometry. Critically, the conserved crossover sequence leading to the RD-domain swap is directly supported by the original cryo-EM density map of ABCA1 and appears to have been overlooked. Our refined ABCA1 model suggests the possibility that ABCA1, despite being an exporter, has highly restrained NBDs that suggest a transport mechanism that is distinct from 'alternating access'.
Assuntos
Transportadores de Cassetes de Ligação de ATPRESUMO
Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane's outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1's outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.
Assuntos
Apolipoproteína A-I , Fosfolipídeos , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Membrana Celular/metabolismo , Lipoproteínas HDL/metabolismo , Fosfolipídeos/metabolismo , Domínios ProteicosRESUMO
The substrate-binding protein YfeA (also known as YPO2439 or y1897) is a polyspecific metal-binding protein that is crucial for nutrient acquisition and virulence in Yersinia pestis, the causative microbe of plague. YfeA folds into a monomeric c-clamp like other substrate-binding proteins and has two metal-binding sites (sites 1 and 2). Site 2 is a bidentate surface site capable of binding Zn and Mn atoms and is a unique feature of YfeA. Occasionally, the site 2 residues of two YfeA molecules will cooperate with the histidine tag of a third YfeA molecule in coordinating the same metal and lead to metal-dependent crystallographic packing. Here, three crystal structures of YfeA are presented at 1.85, 2.05 and 2.25â Å resolution. A comparison of the structures reveals that the metal can be displaced at five different locations ranging from â¼4 to â¼16â Å away from the canonical site 2. These observations reveal different configurations of site 2 that enable cooperative metal binding and demonstrate how site 2 is dynamic and freely available for inter-protein metal coordination.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cloretos/metabolismo , Compostos de Manganês/metabolismo , Yersinia pestis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Conformação ProteicaRESUMO
The multidrug transporter P-glycoprotein (Pgp)/ABCB1/MDR1 plays an important role in multidrug resistance (MDR) and detoxification owing to its ability to efflux an unusually large and chemically diverse set of substrates. Previous phenylalanine-to-alanine scanning mutagenesis of Pgp revealed that nearly all mutations retained full MDR function and still permitted substrate transport. This suggests that either the loss of any single aromatic side chain did not affect the ligand-binding modes or that highly adaptive and compensatory drug recognition is an intrinsic property including ligand-binding shifts that preserve function. To explore this hypothesis, the ATPase function and crystallographic localization of five single-site mutations in which the native aromatic residue directly interacted with the environmental pollutant BDE-100, as shown in previous crystal structures, were tested. Two mutants, Y303A and Y306A, showed strong BDE-100 occupancy at the original site (site 1), but also revealed a novel site 2 located on the opposing pseudo-symmetric half of the drug-binding pocket (DBP). Surprisingly, the F724A mutant structure had no detectable binding in site 1 but exhibited a novel site shifted 11â Å from site 1. ATPase studies revealed shifts in ATPase kinetics for the five mutants, but otherwise indicated a catalytically active transporter that was inhibited by BDE-100, similar to wild-type Pgp. These results emphasize a high degree of compensatory drug recognition in Pgp that is made possible by aromatic amino-acid side chains concentrated in the DBP. Compensatory recognition forms the underpinning of polyspecific drug transport, but also highlights the challenges associated with the design of therapeutics that evade efflux altogether.
RESUMO
Although the 1,2,3-triazole is a commonly used amide bioisostere in medicinal chemistry, the structural implications of this replacement have not been fully studied. Employing X-ray crystallography and computational studies, we report the spatial and electronic consequences of replacing an amide with the triazole in analogues of cystic fibrosis drugs in the VX-770 and VX-809 series. Crystallographic analyses quantify subtle differences in the relative positions and conformational preferences of the R1 and R2 substituents attached to the amide and triazole bioisosteres. Computational studies derived from the X-ray data highlight the improved hydrogen bonding donor and acceptor capabilities of the amide in comparison to the triazole. This analysis of the spatial and electronic differences between the amide and 1,2,3-triazole will inform medicinal chemists as they consider using the triazole as an amide bioisostere.
Assuntos
Amidas/química , Aminofenóis/química , Aminopiridinas/química , Benzodioxóis/química , Fibrose Cística/tratamento farmacológico , Quinolonas/química , Triazóis/química , Amidas/uso terapêutico , Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Quinolonas/uso terapêutico , Triazóis/uso terapêuticoRESUMO
Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/classificação , Domínios Proteicos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Dobramento de ProteínaRESUMO
In the structural biology of bacterial substrate-binding proteins (SBPs), a growing number of comparisons between substrate-bound and substrate-free forms of metal atom-binding (cluster A-I) SBPs have revealed minimal structural differences between forms. These observations contrast with SBPs that bind substrates such as amino acids or nucleic acids and may undergo >60° rigid-body rotations. Substrate transfer in these SBPs is described by a Venus flytrap model, although this model may not apply to all SBPs. In this report, structures are presented of substrate-free (apo) and reconstituted substrate-bound (holo) YfeA, a polyspecific cluster A-I SBP from Yersinia pestis. It is demonstrated that an apo cluster A-I SBP can be purified by fractionation when co-expressed with its cognate transporter, adding an alternative strategy to the mutagenesis or biochemical treatment used to generate other apo cluster A-I SBPs. The apo YfeA structure contains 111 disordered protein atoms in a mobile helix located in the flexible carboxy-terminal lobe. Metal binding triggers a 15-fold reduction in the solvent-accessible surface area of the metal-binding site and reordering of the 111 protein atoms in the mobile helix. The flexible lobe undergoes a 13.6° rigid-body rotation that is driven by a spring-hammer metal-binding mechanism. This asymmetric rigid-body rotation may be unique to metal atom-binding SBPs (i.e. clusters A-I, A-II and D-IV).
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Yersinia pestis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Zinco/metabolismoRESUMO
P-glycoprotein (Pgp) is an ATP-binding cassette transporter that eliminates toxins from the cell but causes multidrug resistance in chemotherapies. The crystal structures of Pgp revealed drug-like compounds bound to an inward-facing conformation in which the energy-harnessing nucleotide binding domains (NBDs) were widely separated with no interfacial interaction. Following drug binding, inward-facing Pgp must transition to an NBD dimer conformation to achieve ATP binding and hydrolysis at canonical sites defined by both halves of the interface. However, given the high degree of flexibility shown for this transporter, it is difficult to envision how NBDs overcome entropic considerations for achieving proper alignment in order to form the canonical ATP binding site. We explored the hypothesis that substrate occupancy of the polyspecific drug-binding cavity plays a role in the proper alignment of NBDs using computational approaches. We conducted twelve atomistic molecular dynamics (MD) simulations (100-300 ns) on inward-facing Pgp in a lipid bilayer with and without small molecule substrates to ascertain effects of drug occupancy on NBD dimerization. Both apo- and drug-occupied simulations showed NBDs approaching each other compared to the crystal structures. Apo-Pgp reached a pseudo-dimerization in which NBD signature motifs for ATP binding exhibited a significant misalignment during closure. In contrast, occupancy of three established substrates positioned by molecular docking achieved NBD alignment that was much more compatible with a canonical NBD dimerization trajectory. Additionally, aromatic amino acids, known to confer the polyspecific drug-binding characteristic of the internal pocket, may also govern polyspecific drug access to the cavity. The enrichment of aromatics comprising the TM4-TM6 portal suggested a preferential pathway over the aromatic-poor TM10-TM12 for lateral drug entry from the lipid bilayer. Our study also suggested that drug polyspecificity is enhanced due to a synergism between multiple drug-domain interactions involving 36 residues identified in TM1, 5, 6, 7, 11 and 12.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Simulação de Dinâmica Molecular , Nucleotídeos/química , Animais , Sítios de Ligação , Bicamadas Lipídicas/química , Camundongos , Simulação de Acoplamento Molecular/métodos , Ligação Proteica , Domínios Proteicos , Multimerização ProteicaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0113069.].
RESUMO
We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation. X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.
Assuntos
Fracionamento Celular/métodos , Bactérias Gram-Negativas/patogenicidade , Metais/química , Radioisótopos/uso terapêutico , Espectrometria por Raios X/métodos , Metais/análiseRESUMO
Biological chelating molecules called siderophores are used to sequester iron and maintain its ferric state. Bacterial substrate-binding proteins (SBPs) bind iron-siderophore complexes and deliver these complexes to ATP-binding cassette (ABC) transporters for import into the cytoplasm, where the iron can be transferred from the siderophore to catalytic enzymes. In Yersinia pestis, the causative agent of plague, the Yersinia iron-uptake (Yiu) ABC transporter has been shown to improve iron acquisition under iron-chelated conditions. The Yiu transporter has been proposed to be an iron-siderophore transporter; however, the precise siderophore substrate is unknown. Therefore, the precise role of the Yiu transporter in Y. pestis survival remains uncharacterized. To better understand the function of the Yiu transporter, the crystal structure of YiuA (YPO1310/y2875), an SBP which functions to present the iron-siderophore substrate to the transporter for import into the cytoplasm, was determined. The 2.20 and 1.77â Å resolution X-ray crystal structures reveal a basic triad binding motif at the YiuA canonical substrate-binding site, indicative of a metal-chelate binding site. Structural alignment and computational docking studies support the function of YiuA in binding chelated metal. Additionally, YiuA contains two mobile helices, helix 5 and helix 10, that undergo 2-3â Å shifts across crystal forms and demonstrate structural breathing of the c-clamp architecture. The flexibility in both c-clamp lobes suggest that YiuA substrate transfer resembles the Venus flytrap mechanism that has been proposed for other SBPs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Chaperonas Moleculares/química , Sideróforos/metabolismo , Yersinia pestis/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Ferro/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação ProteicaRESUMO
Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading.
Assuntos
Metais/metabolismo , Proteínas Periplásmicas de Ligação/química , Peste/microbiologia , Fatores de Virulência/química , Yersinia pestis/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ferro/metabolismo , Manganês/metabolismo , Modelos Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Fatores de Virulência/metabolismo , Yersinia pestis/metabolismo , Zinco/metabolismoRESUMO
Protein, peptides, and nucleic acids are biomolecules that drive biological processes in living organisms. An enormous amount of structural data for a large number of these biomolecules has been described with atomic precision in the form of structural "snapshots" that are freely available in public repositories. These snapshots can help explain how the biomolecules function, the nature of interactions between multi-molecular complexes, and even how small-molecule drugs can modulate the biomolecules for clinical benefits. Furthermore, these structural snapshots serve as inputs for sophisticated computer simulations to turn the biomolecules into moving, "breathing" molecular machines for understanding their dynamic properties in real-time computer simulations. In order for the researcher to take advantage of such a wealth of structural data, it is necessary to gain competency in the use of computer molecular visualization tools for exploring the structures and visualizing three-dimensional spatial representations. Here, we present protocols for using two common visualization tools--the Web-based Jmol and the stand-alone PyMOL package--as well as a few examples of other popular tools.