Damien Jefferies

Interaction of the Antimicrobial Peptide Polymyxin B1 with Both Membranes of E. coli: A Molecular Dynamics Study

Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.

An Accurate In Vitro Model of the E. coli Envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir–Blodgett and Langmuir–Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

Through the Lipopolysaccharide Glass: A Potent Antimicrobial Peptide Induces Phase Changes in Membranes

In the following, molecular simulations are used to reveal unexpected behavior within bacterial membranes. We show that lipopolysaccharide molecules found in these membranes form viscous amorphous solids when they are interlinked with monovalent and divalent cations. The bilayers exhibit both liquid and glassy characteristics, due to the coexistence of both liquid and crystalline domains in the bilayer. Polymyxin B1, a potent antimicrobial peptide, is shown to increase order within the lipopolysaccharide bilayers by inducing the formation of crystalline patches. Crucially we are able to decompose the energetics of insertion into their enthalpic and entropic components. The present coarse-grain molecular dynamics study provides unprecedented insights into the antibacterial action of antimicrobial peptides, thus paving the way for development of novel therapeutic agents to treat multiple drug resistant Gram-negative bacteria.

The outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB and OmpX have unique lipopolysaccharide fingerprints.

The outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and in the outer leaflet they contain almost exclusively lipopolysaccharide (LPS) molecules. In E. coli, the outer membrane contains a wide range proteins with a beta barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of twenty-two strands. Here we report coarse-grain molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF and EstA in a range of membrane environments, which are representative of the in vivo for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 microseconds of simulation for each protein. Author summary We present data from over 200 microseconds of coarse-grain simulations that show the complexities of protein-lipid interactions within the outer membranes of Gram-negative bacteria. We show that the slow movement of lipolysaccharide molecules necessitate simulations of over 30 microsecond duration to achieve converged properties such as protein tilt angle. Each of the six proteins studied here shows a unique pattern of interactions with the outer membrane and thus constitute a ‘fingerprint’ or ‘signature’.