Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment

Abstract

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain’s membrane association. Coarse-grained molecular dynamics (CG-MD) simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8-TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.

Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers

“…Two distinctive membrane-bound conformations were identified in extensive equilibrium unrestrained simulations of the protein with the membrane, and characterized using umbrella sampling (US) simulations. 22 Both predicted membrane-bound conformations showed helices TH8–TH9 with a near parallel orientation relative to the membrane plane, which was in agreement with low resolution information reported by solid state nuclear magnetic resonance (NMR). 11 However, only limited characterization of the preferred modes of the protein binding with the lipid is possible using the coarse-grained approaches.…”

“…19 The model of the partially unfolded state resulted from such MD simulations is characterized by exposed hydrophobic and charged sites on the protein surface that were assumed to facilitate subsequent membrane binding. 19,22 Furthermore, the coarse-grained MD simulations suggested that the partially unfolded model of the T-domain initially associates to the membrane through two distinctive conformations (B1 and B2). 22 In this study, atomistic MD simulations at T = 310 K showed that conformations B1 and B2 retained their overall structure, orientation, and membrane contacts over several hundreds of nanoseconds.…”

“…22 In this study, a representative snapshot from each bound conformation was extracted from an umbrella window simulation corresponding to the lowest free energy (see 22 ). Each coarse-grained model of the T-domain bound to a bilayer was converted into an all-atom model using the conversion protocol of Cojocaru et al 43 The original number of phospholipids in the coarse-grained model was 512, which was reduced to 192 lipids in the atomistic model.…”

“…22 Using a coarse-grained model allowed us to perform free unrestrained simulations of the T-domain association with membranes and to demonstrate that presence of anionic lipid in the membrane increased the lifetime of the protein–membrane complexes. Two distinctive membrane-bound conformations were identified in extensive equilibrium unrestrained simulations of the protein with the membrane, and characterized using umbrella sampling (US) simulations.…”

Membrane Protein Folding & Lipid Interactions: Theory & Experiment

“…The folding of transmembrane α-helices, which is of fundamental importance for membrane protein assembly, was studied in the context of both lipid bilayer [6] and translocon-like pores [7]. Several studies demonstrated how computational and experimental approaches can be integrated to gain insights into the mechanism of interfacial interactions of diphtheria toxin [8] and to validate spectroscopic tools for determining membrane penetration [9]. The significance of computational studies is further highlighted by two reviews, one specifically dedicated to describing molecular motions associated with the functioning of the voltage-sensing domains [10], while the other is dedicated to reviewing multiple applications of a novel membrane representation termed highly mobile membrane mimetic (HMMM), resulting in an efficient sampling of lipid–protein interactions at atomic resolution [11].…”