Design of antimicrobial peptides

Background: A ubiquitous mechanism by which multidrug resistance is conferred to bacteria involves the active efflux of a broad variety of cytotoxic compounds by transmembrane multidrug resistance proteins. The small multidrug resistance (SMR) family of membrane-bound transporters is ubiquitous in bacteria, including the pathogens L. pneumoniae, M. tuberculosis, P. aeruginosa, B. pertussis, N. meningitis, S. aureus has received less attention than other transporter families because a high-resolution structure has not been available.

SMR proteins use the electrochemical potential of proton  influx to actively pump toxic compounds from the cytoplasm, causing resistance to a wide variety of quarternary ammonium compounds some of which are used as disinfectants  (e.g. benzalkonium chloride), and also promote resistance to antibiotics such as ampicillin, erythromycin and tetracycline.

Results: We used computer simulations to refine a coarse-grained crystal structure of E. coli EmR, and to design stapled peptide inhibitors, which were validated in vitro using a fluorescence-based Ethidium bromide efflux assay.  Specifically, two of the four top-scoring peptides selected for experimental testing caused significantly increased accumulation of Ethidium and growth inhibition in live cells without nonspecific toxicity.  In the future, we plan to design consensus inhibitors with broad efficacy against multiple bacteria. 

Refined structure of EmrE. (A and B) Side views showing monomer chains 1 (yellow) and 2 (blue), with helices H4 of both monomers drawn in purple. (C) Side view showing the dimer interface and TPP inside the binding pocket; the orientation in C is obtained from those (A and B) by 90 deg and –90 deg rotations, respectively, about the vertical axis using the right-hand rule. The refined structure in ribbon representation is superimposed on the Cα -only PDB structure, drawn as connected cylinders. (D) Side view showing the solvation environment of the dimer, with the lipids with any atom within 2.7 Å of the protein backbone outlined in dark blue and water oxygens within 5 Å of a protein atom drawn as small green spheres. (E) RMSD between the evolving simulation structure and the initial minimized structure. Only the helix backbones were used for the calculation. (F) RMSFs computed from MD with those obtained from B-factors in the PDB; for each residue, the RMSF shown represents an average over the coordinates of the residue heavy atoms; a 3-point smoothing filter was applied to the simulation and PDB data.