RESUMO
The OccK protein subfamily located in the outer membrane of Pseudomonas aeruginosa contains dynamic channels with several conformational states that range from open to closed forms. The molecular determinants of the OccK channels that contribute to the diverse gating have, however, remained elusive so far. Performing molecular dynamics (MD) simulations on OccK5 (OpdH) as an example, local fluctuations of loop L7 mediated by a single residue were identified that effectively gate the channel. The features of this gate residue were studied by single-channel electrophysiology and site-directed mutagenesis demonstrating that this gate residue indeed confers unique gating properties to the OccK channels. In support of these functional measurements, MD simulations highlight the correlations between the size of the side-chain belonging to the gate residue on one side and the pore size as well as the L7 flexibility on the other side.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Canais Iônicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/químicaRESUMO
The Gram-negative organism Campylobacter jejuni is the major cause of food poisoning. Unlike Escherichia coli, which has two major porins, OmpC and OmpF, C. jejuni has one, termed major outer membrane protein (MOMP) through which nutrients and antibiotics transit. We report the 2.1-Å crystal structure of C. jejuni MOMP expressed in E. coli and a lower resolution but otherwise identical structure purified directly from C. jejuni. The 2.1-Å resolution structure of recombinant MOMP showed that although the protein has timeric arrangement similar to OmpC, it is an 18-stranded, not 16-stranded, ß-barrel. The structure has identified a Ca2+ bound at the constriction zone, which is functionally significant as suggested by molecular dynamics and single-channel experiments. The water-filled channel of MOMP has a narrow constriction zone, and single-molecule studies show a monomeric conductivity of 0.7±0.2 nS and a trimeric conductance of 2.2±0.2 nS. The ion neutralizes negative charges at the constriction zone, reducing the transverse electric field and reversing ion selectivity. Modeling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the presence metal ion.
