Theoretical analysis of ion conductance and gating transitions in the OpdK (OccK1) channel

Pothula, Karunakar R.; Kleinekathöfer, Ulrich

doi:10.1039/c5an00036j

Cited by 13 publications

(8 citation statements)

References 50 publications

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“…S10 ). However, our mechanism should not be generalized to porins with multiple internal loops, such as the OccK and OccD family in Pseudomonas aeruginosa , since the open–closed transitions of these porins might involve the motion of multiple loops ( 50 , 51 ).…”

Section: Resultsmentioning

confidence: 99%

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

Vasan

Haloi

Ulrich

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Vasan

Haloi

Ulrich

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Differently from general channels, the eyelet region is lined by two long flexible loops that seem to work as a gate, as recently shown for OpdK/OccK1. [85] More interestingly, the dynamics of the two loops can create two alternative paths in OprD/OccD1. [50] Substrates appeared to use a different path depending on their charge distribution and polarity.…”

Section: Specific Porins As Evolution Of General Porinsmentioning

confidence: 99%

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

Scorciapino

Acosta‐Gutiérrez

Benkerrou

et al. 2017

J. Phys.: Condens. Matter

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The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI-Translocation consortium. The synergistic combination of structural data, in vitro assays and computer simulations has proven to give new insights towards the identification and description of physico-chemical properties modulating permeation. Once similar general rules are identified, we believe that the use of virtual screening techniques will be very helpful in searching for new molecular scaffolds with enhanced permeation, and that molecular modeling will be of fundamental assistance to the optimization stage.

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“…One challenging prerequisite for using these monomeric β-barrels in single-molecule detection is obtaining a quiet single-channel electrical signature that is freed of current gating fluctuations [13], otherwise interfering with the analyte-induced current blockades resulted from single-molecule detection. Spontaneous fluctuations in β-barrel protein channels, pores, and porins have been extensively explored [7, 14–21]. In general, these fluctuations are produced by conformational alterations of the large extracellular loops, which many times permanently or transiently fold back into the pore interior [7, 16].…”

Section: Introductionmentioning

confidence: 99%

Global redesign of a native β-barrel scaffold

Wolfe

Mohammad

Thakur

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded β-barrel protein into a large-conductance protein pore. Here, we show that this redesigned β-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric β-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

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Theoretical analysis of ion conductance and gating transitions in the OpdK (OccK1) channel

Cited by 13 publications

References 50 publications

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

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

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

Global redesign of a native β-barrel scaffold

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