Abstract
The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the β-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in porin channels.
Highlights
All biological cells are enclosed by lipid bilayers
We demonstrate that a combination of high-resolution single-channel electrophysiology, novel high-sensitivity electrophysiology analysis methods[23], and atomistic molecular dynamics simulations allowed us to reveal in structural detail a drug-channel binding interaction that is too weak to be captured by X-ray crystallography
The binding site revealed by our approach is wedged between the clusters of conserved acidic and basic residues of the eyelet region, suggesting a common binding site for zwitterionic drugs within porin channels, in which ligand binding is strongly determined by the transverse electrostatic field[33,44,49]
Summary
All biological cells are enclosed by lipid bilayers. The translocation of hydrophilic molecules, such as nutrients and metabolic waste products, across these membranes is often rate-limiting for cellular growth and survival. Antibiotics with activity against Gram-negative bacteria must be capable of permeating porin channels, which are located in the outer membrane of their cell envelope[5]. This requirement imposes tight constraints on the physicochemical properties of permeants, and enables pathogenic bacteria to evolve mutated porins, incurring a substantial decrease or complete loss of antibiotic uptake rates[6,7,8,9]. In order to provide structural information at sufficient detail for improved drug design[20,33], it is important to reliably identify these scenarios, and to characterise the molecular determinants of any potential binding event between the antibiotic molecule and the protein channel[34,35]. We demonstrate how we have combined new and traditional methodologies of membrane channel characterisation to achieve this
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