Role of internal loop dynamics in antibiotic permeability of outer membrane porins

Abstract

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open–closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF β-barrel. Mutation of key residues important for the loop’s conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating

“…Possibly, the partially closed states correspond to intermediate size fluctuations of the current in single channel reading, while the closed state corresponds to short-lived and infrequent large amplitude fluctuations. Recently, a detailed Markov state model (MSM) study on OmpF reported large-scale backbone conformational changes in the L3 loop involved in the open to closed transition . A two-step mechanism was reported wherein E117 at the L3 tip interacts with the R42 on the opposite wall, followed by interactions of D121 with R132 and R82 leading to pore closure.…”

“…This present results are consistent with previous experimental results on L3 loop tethering in ref , wherein two of the double mutants investigated (E117C–A333C and E117C–D312C) did not show any impairment of the voltage gating as compared to wild-type OmpF, indicating that the L3 tip at residue E117 remains stable in the event of a voltage-mediated pore closure. Interestingly, the MSM investigation by Vasan et al also reports an almost identical structural transition that involves motion of the D121 residue and its interaction with the R82 residue with a stable L3 tip as observed in the present study, albeit with a much lower likelihood.…”

“…Recently, a detailed Markov state model (MSM) study on OmpF reported large-scale backbone conformational changes in the L3 loop involved in the open to closed transition. 65 A two-step mechanism was reported wherein E117 at the L3 tip interacts with the R42 on the opposite wall, followed by interactions of D121 with R132 and R82 leading to pore closure. Although the study focuses particularly on the spontaneous gating mechanisms, that is, open to close transitions at low applied voltages, the results also highlight the role of loop L3 conformational dynamics.…”

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance

“…Protein modelling of the WT allele show that the NTC domain extends from the periplasm inwards into the porin and interacts with the inner loop 3 (L3), which traverses the upper region of the OmpU towards to the extracellular side. Domains analogous to NTC and L3 in other porins contribute to pore size and altered open and close conformation states [37, 62-67]. Given our findings, it is likely that the presence of the N-terminal coil contributes to a smaller pore size [37] in the WT allele leading to exclusion of bile molecules in contrast to the sensitive alleles.…”

Review on Characterization, Properties, and Analytical Methods of Cefepime

“…However, unlike other third-generation cephalosporins, CP possesses a cephem nucleus substituted with a positively charged NMR, making it a zwitterion [ 2 ]. This zwitterionic property permits penetration of CP to Gram (+) bacteria's porin channels rapidly [ 10 , 11 ]. CP is used effectively to treat severe urinary and respiratory tract infections, as well as infections of the skin, soft tissues, and the women's reproductive tract among patients with febrile neutropenia.…”