Structure and Membrane Topology of the Pore-Forming Peptide Maculatin 1.1

Abstract

Antimicrobial peptides (AMP) that target membranes are an attractive alternative to classic antibiotics, since they do not require internalization nor target a specific stereo-structure, thus limiting development of bacterial resistance. Their mode of action involves the disruption of lipid membranes; however, the relationship between lipid membrane structure and peptide potency remains unclear. We present the structural investigation of the AMP maculatin 1.1 (Mac1) in DPC micelles and DHPC/DMPC isotropic (q= 0.5) bicelles. Using solution and solid-state NMR with paramagnetic relaxation enhancement agents (PRE) and molecular dynamics (MD), we demonstrate the important role of the membrane structure in modulating the structure and location of Mac1. HSQC of specifically 15N labeled Mac1 in buffer displayed a narrow chemical shift dispersion that is typical of random coil structures. Introduction of micelles and bicelles produced chemical shift dispersions characteristic of helical structures, with differences suggesting that Mac1 adopts a different degree of helicity dependent on the curvature. 3D TROSY-NOESY allowed assignment of the sequential 15N labeled residues, and determination of a 3D helical structure in phospholipid micelles and bicelles, the latter producing the greatest helical stretch. Titration of the PRE agent Gd3+-(DTPA) showed that the central core of Mac1 is protected in bicelles while in micelles only the N-term is exposed to the PRE effect. MD simulations in DPC micelles revealed N-term exposure to the solvent, and they also suggested that Mac1 bent to adapt the curved micelle structure. Experiments will be repeated with 4 and 8 peptides per micelles.