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δ-lysin, secreted by a Gram-positive bacterium Staphylococcus aureus, is a 26-residue membrane active peptide that shares many common features with antimicrobial peptides (AMPs). However, it possesses a few unique features that differentiate itself from typical AMPs. In particular, δ-lysin has zero net charge, even though it has many charged residues, and it preferentially lyses eukaryotic cells over bacterial cells. Here, we present the results of coarse-grained molecular dynamics simulations of δ-lysin interacting with a zwitterionic membrane over a wide range of peptide concentrations. When the peptides concentration is low, spontaneous dimerization of peptides is observed on the membrane surface, but deep insertion of peptides or pore formation was not observed. However, the calculated free energy of peptide insertion suggests that a small fraction of peptides is likely to be present inside the membrane at the peptide concentrations typically seen in dye efflux experiments. When the simulations with multiple peptides are carried out with a single pre-inserted transmembrane peptide, spontaneous pore formation occurs with a peptide-to-lipid ratio (P/L) as low as P/L = 1:42. Inter-peptide salt bridges among the transmembrane peptides seem to play a role in creating compact pores with very low level of hydration. More importantly, the transmembrane peptides making up the pore are constantly pushed to the opposite side of the membrane when the mass imbalance between the two sides of membrane is significant. Thus, the pore is very dynamic, allowing multiple peptides to translocate across the membrane simultaneously.

The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic ‘aqueous’ H+ conductance (GAQ). Mutation of a highly conserved ‘gating charge’ residue in the S4 helix (R1H) confers a resting-state H+ ‘shuttle’ conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1. DOI: http://dx.doi.org/10.7554/eLife.18017.001

The structure of an Hv1-based chimeric voltage sensor protein (mHv1cc) was recently solved by X-ray crystallography. Although mHv1cc mediates voltage-gated H+ currents when expressed in mammalian cells, the crystallized protein likely represents a closed, possibly resting, conformation. However, the mHv1cc structure is apparently incompatible with our experimental measurements of the resting-state H+ ‘shuttle’ conductance (GSH) in hHv1 R205H. In order to explore possible conformations of the Hv1 voltage sensor (VS) domain in its resting state, we created an Hv1 VS domain homology model (Hv1 D) based on a previously reported resting-state Rosetta model structure of the Shaker K+ channel VS domain (Pathak, et al. 2007) and subjected the Hv1 model to all-atom MD simulations. We subsequently created a model of the Hv1 D R205H point mutant that mediates GSH. In contrast to mHv1cc and a resting-state model of the Ciona Hv1 VS domain (Chamberlin, et al. 2013), the central crevice in our new model is not obviously occluded by hydrophobic side chains. A prominent feature of Hv1 D R205H is that the imidazole ring of the introduced histidine appears to be simultaneously accessible to intra- and extra-cellular aqueous vestiblues, and therefore appears competent for Grotthuss-type proton conduction. Hv1 D represents the first VS domain resting-state model structure that satisfies the rigorous structural constraints imposed by experimental data and thus serves as a template for understanding the structural basis of VS activation in a variety of VS domain-containing proteins.