Voltage-dependent structural changes of the membrane-bound anion channel hVDAC1 probed by SEIRA and electrochemical impedance spectroscopy

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The voltage-dependent anion channel (VDAC) is a transmembrane protein that regulates the transfer of metabolites between the cytosol and the mitochondrium. Opening and partial closing of the channel is known to be driven by the transmembrane potential viaa mechanism that is not fully understood. In this work, we employed a spectroelectrochemical approach to probe the voltage-induced molecular structure changes of human VDAC1 (hVDAC1) embedded in a tethered bilayer lipid membrane on a nanostructured Au electrode. The model membrane consisted of a mixed self-assembled monolayer of 6-mercaptohexanol and (cholesterylpolyethylenoxy)thiol, followed by the deposition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles including hVDAC1. The stepwise assembly of the model membrane and the incorporation of hVDAC1 were monitored by surface enhanced infrared absorption and electrochemical impedance spectroscopy. Difference spectra allowed for identifying the spectral changes which may be associated with the transition from the open to the “closed” states by shifting the potential above or below the transmembrane potential determined to beca.0.0 Vvs.the open circuit potential. These spectral changes were interpreted on the basis of the orientation- and distance-dependent IR enhancement and indicate alterations of the inclination angle of the β-strands as crucial molecular events, reflecting an expansion or contraction of the β-barrel pore. These protein structural changes that do not confirm nor exclude the reorientation of the α-helix are either directly induced by the electric field or a consequence of a potential-dependent repulsion or attraction of the bilayer.DFG, EXC 314, Unifying Concepts in CatalysisDFG, SFB 803, Funktionalität kontrolliert durch Organisation in und zwischen Membrane

Electrophysilogical characterization of the mitochondrial porin VDAC1 and the antimicrobial peptide Dermcidin in solvent-free model membranes

Solvent free lipid bilayers were used for the electrophysiological characterization of the most abundant protein of the outer mitochondrial membrane, the isoform 1 of the voltage dependent anion channel (VDAC1), and the antimicrobial peptide Dermcidin. The bilayers were prepared via spreading of giant unilamellar vesicles (GUVs) over a single pore in a borosilicate glass surface. To gain deeper insight in its gating behavior, wild type VDAC1 as well as two VDAC1 mutants were incorporated into GUVs which then were spread over the aperture. Subsequent analysis of single channels at varying transmembrane potentials Um revealed the transition between an open conformation with a conductance of Go = 4.0 nS at low potentials and a closed conformation with Gc = 2.0 nS in 1 M KCl for Um ≥ 30 mV and Um ≤ 30 mV for all VDAC1 variants. This gating between the main VDAC1 states with low transition rates of kmg = 0.1 2 s-1 did not necessarily occur at high transmembrane potentials as the open probability Po did not fall below 90 % even at Um ≥ 30 mV on a single channel level. Furthermore, a VDAC1 mutant with three additional amino acids at its N-terminal end (RGS VDAC1) exhibited a previously not characterized second gating that superimposed the main gating of VDAC1. Closer analysis of this fast gating resulted in a lower amplitude of ΔGfg = 0.5 1.2 nS and much higher transition rates of kfg = 18 911 s 1 with higher rates at low voltages and vice versa. A second mutant (V17C/A205C VDAC1) contained an artificially introduced disulfide bond that prevented the movement of a certain protein structure as putative molecular basis of the VDAC1 conductance transition. However, V17C/A205C VDAC1 did not exhibit an altered behavior compared to the wild type VDAC1. The antimicrobial activity of Dermcidin (DCD) mainly consists of its ability to perforate lipid bilayers. To elucidate the postulated zinc dependency of the DCD activity, stable membranes were prepared and subsequently incubated with DCD in a zinc free environment or in the presence of Zn2+ ions. High DCD activity was detected in the presence of Zn2+ ions whereas no activity was found under zinc free conditions or in the presence of Mg2+ ions. These findings and the inactivation of DCD via the alteration of a zinc binding site in a mutant (H38A DCD) proved the zinc dependency of the DCD activity

Crystal structure and functional mechanism of a human antimicrobial membrane channel

Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These host-defense peptides compromise the integrity of microbial cell membranes and thus evade pathways by which bacteria develop rapid antibiotic resistance. Although more than 1,700 host-defense peptides have been identified, the structural and mechanistic basis of their action remains speculative. This impedes the desired rational development of these agents into next-generation antibiotics. We present the X-ray crystal structure as well as solid-state NMR spectroscopy, electrophysiology, and MD simulations of human dermcidin in membranes that reveal the antibiotic mechanism of this major human antimicrobial, found to suppress Staphylococcus aureus growth on the epidermal surface. Dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidate the unusual membrane permeation pathway for ions and show adjustment of the pore to various membranes. Our study unravels the comprehensive mechanism for the membrane-disruptive action of this mammalian host-defense peptide at atomistic level. The results may form a foundation for the structure-based design of peptide antibiotics

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

International audienceThe antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1.

The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.peerReviewe