NMR characterization of the voltage-gated proton channel Hv1

Getting ions across the membrane barrier is one of the most fundamental processes in life. Ion permeation of membranes is catalyzed by membrane-embedded ion channels and transporters. Voltage-gated ion channels are activated by changes in membrane potential. Voltage-sensing is achieved by a protein domain the so-called voltage-sensing domain (VSD). In response to voltage change, the helices of the VSD move relative to the lipid bilayer this conformational change results in the opening of a selective ion pore, which spans the cell membrane. This allows the cell to move ions across the lipid bilayer. These proteins are required among others for the generation and propagation of nerve impulses, proton extrusion from the cell and muscle contraction. The first gene of a voltage-gated proton channel was discovered in 2006. It consists of a VSD and a C-terminal dimerization domain, however, it lacks a separate ion-conducting pore that is present in the voltage-gated Na+ , Ca2+ and K+ channels. The full-length channel acts as a dimer, however truncation of the cytoplasmic C-terminus leads to a fully functional monomeric voltage-gated proton channel, Hv1M. Proton conduction through voltage- gated H+ channels is not only voltage dependent, it is also affected by the pH gradient across the cell membrane. Conformational changes are essential for the voltage-gating and the pH-sensing mechanism of voltage-gated proton channel Hv1. NMR is unique in its ability to probe structural plasticity of proteins at atomic resolution on a wide range of times-scales. In this project we will determine the solution structures of the open and closed state of Hv1M. In addition to providing the atomic coordinates of this domain, we will characterize the different motional states of this molecule over a broad range of time-scales using NMR relaxation experiments, for understanding the intrinsic dynamics of Hv1M that is coupled to voltage sensing and proton conduction. We anticipate that our study will provide a genuine atomistic description of the pH-dependent voltage-gated proton conduction mechanism of Hv1M, and will significantly contribute to the understanding of voltage-gated proton channels.

In this project of the Austrian Science Fund FWF we characterized the substrate recognition and substrate transport by the mitochondrial ADP/ATP carrier (AAC). AAC is part of the mitochondrial carrier family, which is a family of transporters that catalyze the trafficking of metabolites, nucleotides, ions and vitamins across the mitochondrial inner membrane. Over 40 different carrier proteins have been identified in human, and most of them are linked to diseases characterized by metabolic dysfunction or defective energy production. Mitochondrial carriers are of academic interest as well, because unlike the 2-fold pseudo symmetry of most solute transporters, they adopt a 3-fold pseudo symmetry. Using nuclear magnetic resonance (NMR) relaxation techniques we were able to show that AAC interconverts between two different conformations, which are crucial for substrate transport, in the absence of substrate. In the presence of substrate the rate of interconversion increases by a factor of two. Binding of the inhibitor CATR to AAC decreases the rate of conformational change. For another carrier, the short Ca2+- binding mitochondrial carrier (SCaMC), which adopts an extramembrane N- terminal domain (NTD) for ATP transport regulation, we showed that NTD undergoes a structural change triggered by Ca2+ binding and that this conformational switch directly impacts its ability to interact with the transmembrane region. In the absence of Ca2+, NTD is partially unfolded and interacts specifically with the transmembrane domain (TMD), whereas in the presence of Ca2+ the NTD folds into a compact structure, which abolishes binding to the TMD. Our data suggests a capping mechanism for the Ca2+ dependent regulation of ATP transport of SCaMC in which the apo NTD caps the TMD channel and blocks substrate transport. Binding of Ca2+ to the NTD uncaps it from the TMD and the substrate can be transported. For the voltage- gated proton channel, Hv1, we aimed to characterize the structural and dynamics changes, which occur during proton transport. Hv1 is required for the production of high superoxide concentrations by phagocytes to eliminate pathogens. Hv1 is further expressed in human sperm and is assumed to regulate motility through alkalization- activated calcium channels. We were able to establish an NMR system of Hv1 that allowed us to determine structural restraints and dynamics parameters. The dynamics parameters will allow us to understand the intrinsic dynamics that is coupled to voltage sensing and proton conduction. The collected distance restraints will be employed in an ab initio structure calculation.