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1.
Nature ; 580(7805): 658-662, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32350467

RESUMO

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.


Assuntos
Pseudomonas aeruginosa , Piocinas/química , Piocinas/metabolismo , Bacteriófago T4/química , Bacteriófago T4/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Genes Bacterianos/genética , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especificidade por Substrato , Sistemas de Secreção Tipo VI/química , Sistemas de Secreção Tipo VI/metabolismo
2.
Nature ; 500(7462): 350-353, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23925114

RESUMO

The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Repetições de Microssatélites/fisiologia , Acinetobacter/genética , Acinetobacter/metabolismo , Ligação Proteica , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
3.
J Am Chem Soc ; 135(17): 6562-9, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23560397

RESUMO

Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format X(m)CX(n)CX(o)CX(p) are oxidized to connect two pairs of cysteines (C). This allows the generation of 3 × (m + n + o + p) different peptide topologies because the fourth cysteine can appear in any of the (m + n + o + p) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities.


Assuntos
Compostos Bicíclicos com Pontes/química , Cisteína/química , Peptídeos/química , Alquilação , Sequência de Aminoácidos , Aminoácidos/química , Técnicas de Química Combinatória , Cristalização , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Biblioteca de Peptídeos , Difração de Raios X
4.
Chembiochem ; 14(11): 1316-22, 2013 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-23828687

RESUMO

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.


Assuntos
Aminoácidos/metabolismo , Glicina/metabolismo , Peptídeos/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Bases de Dados de Proteínas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Estabilidade Proteica , Proteólise , Especificidade por Substrato , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
5.
J Mol Biol ; 431(19): 3718-3739, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31325442

RESUMO

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.


Assuntos
Bacteriófagos/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Cristalografia por Raios X , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Especificidade de Hospedeiro , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise , Salmonella enterica/virologia , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato
6.
Viruses ; 10(8)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110933

RESUMO

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to "simple" contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains-two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.


Assuntos
Pseudomonas aeruginosa/química , Piocinas/química , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cátions , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ferro/química , Ferro/metabolismo , Magnésio/química , Magnésio/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sódio/química , Sódio/metabolismo , Especificidade por Substrato , Termodinâmica
7.
Viruses ; 7(8): 4676-706, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26295253

RESUMO

Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender ß-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded ß-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 ß-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the ß-helix is small. It is located near the C-terminal end of the ß-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the ß-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete ß-helix.


Assuntos
Bacteriófago T4/química , Proteínas Virais/química , Bacteriófago T4/metabolismo , Fenômenos Biofísicos , Cristalografia por Raios X , Ácidos Graxos/análise , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
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