RESUMO
Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self-immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward-occluded and a new nucleotide-bound state, high-energy outward-occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross-linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi-drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic-level build-up.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Transporte ProteicoRESUMO
Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors' knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3-6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Bacteriocinas , Proteínas de Escherichia coli/química , Escherichia coli/química , Transportadores de Cassetes de Ligação de ATP/genética , Substituição de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide what is to our knowledge the first structural evidence on the recognition mechanism, and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials.
Assuntos
Anti-Infecciosos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/metabolismo , Receptores de Superfície Celular/metabolismo , Anti-Infecciosos/farmacologia , Endocitose , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Microcins are gene-encoded antibacterial peptides of low molecular mass (<10 kDa), produced by Enterobactericeae. They are produced and secreted under conditions of limited essential nutrients and are active against related species. Bacterial strains under starvation conditions can produce and release microcins that can kill microcin-sensitive cells and therefore have more nutrients for survival. The outer-membrane protein OmpF and FhuA TonB-dependent pathways facilitate the internalization of the MccB17 and MccJ25 microcins into the target cell respectively. The inner-membrane protein SbmA transports the microcins through the inner membrane to the cytoplasmic face. Inside the cell, MccB17 targets DNA gyrase, whereas MccJ25 inhibits the bacterial RNA polymerase.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/fisiologia , Porinas/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Bacteriocinas/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/fisiologia , Modelos Moleculares , Porinas/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Transporte ProteicoRESUMO
Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivo S. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.
RESUMO
Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.