The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the channel known to guide the metabolic flux across the MOM and plays a key role in mitochondrially induced apoptosis. Here, we present the 3D structure of human VDAC1, which was solved conjointly by NMR spectroscopy and x-ray crystallography. Human VDAC1 (hVDAC1) adopts a ␤-barrel architecture composed of 19 ␤-strands with an ␣-helix located horizontally midway within the pore. Bioinformatic analysis indicates that this channel architecture is common to all VDAC proteins and is adopted by the general import pore TOM40 of mammals, which is also located in the MOM.T he outer membrane of mitochondria (MOM) contains three integral membrane protein families, two of which form channels as part of larger protein complexes (for review, see ref. 1). These two MOM complexes, the general import pore TOM and the SAM insertase, allow for the entire translocation and insertion of nearly all newly synthesized proteins destined to the mitochondrial organelle (2, 3). The third protein family of typically high abundance (Ϸ10,000 copies per mitochondrion) is termed voltage-dependent anion channels (VDACs), because of the voltage sensitivity of its open probability (4, 5). Together, this small number of protein families is sufficient for full communication between mitochondria with their cellular environment (1).The VDAC channel was initially described as being reminiscent of bacterial porins and primarily responsible for the exchange of chemical energy equivalents between the cytosol and the mitochondrion (4, 6). Indeed, a variety of structural features (like barrel geometry and dimension) known from the bacterial precursors are maintained (7,8). By contrast, a variety of functions have been ascribed to the VDAC isoforms among which the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol seems to be the most general function (9). The structure of VDAC is of interest because of a substantial body of evidence connecting VDAC to apoptosis. It is suggested that VDAC is a critical player in the release of apoptogenic factors from mitochondria of mammalian cells, and consequently several hypotheses describing the mechanism of mitochondria-mediated apoptosis involving VDAC have been proposed (for review, see ref. 10). Results and DiscussionStructure Determination of hVDAC1: Combining NMR Spectroscopy and X-Ray Crystallography. In a parallel structural biology approach, we set out to characterize the structure of hVDAC1, the major isoform of this channel in mammalian tissues, by a combination of NMR spectroscopy and x-ray crystallography. The idea behind this project was to gain complementary structural information to have a solid basis for future studies, e.g., analysis of protein heterocomplex formation by NMR and crystal structures as a basis for drug target design. Only information derived from both methods and the application of an iterative s...

Although nearly half of today’s major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.Kunitz domain proteins, like the bovine pancreatic trypsin inhibitor (BPTI) 1 (1) or the dendrotoxins (2) are small, basic proteins that contain three highly conserved disulfide bonds. The three disulfide cross-links make these extracellular proteins extremely stable. Two different general functions are known for the different Kunitz proteins. One group, including BPTI, consists of potent protease inhibitors. The complex of BPTI and trypsin is exceptionally stable, with an association constant Ͼ10 MϪ1 (3). The dendrotoxins belong to another group of Kunitz peptides found in the venom of the black mamba, which block different potassium channels with a high degree of specificity and selectivity (4). In snake and scorpion venoms a diverse set of different potassium channel blockers have been characterized (2).Despite the great variety of toxins from the venoms of the predatory cone snails, relatively few have been identified so far that interact with potassium channels (5). Most conotoxins are small, peptidic toxins that typically contain 10 -30 amino acids and bind with high affinity and specificity to various ligand-or voltage-gated ion channels. One striking feature of these peptides is that they usually contain a diverse complement of posttranslational modifications, like C-terminal amidation, hydroxyprolines, or glycosylation of serine or threonine (6). Conotoxins can be broadly divided into two groups, the non-disulfide-rich peptides and the disulfide-rich conotoxins. The latter conotoxins are further separated into several families based on cysteine bridge pattern and biological activities (5).The potassium channel-targeted toxin conkunitzin-S1 (Conk-S1) from the venom of Conus striatus is the first member of a new family of polypeptides. Recently it has been shown that Conk-S1 blocks Shaker potassium channels with an IC 50 of less than 100 nM.2 Compared with most toxins from Conus venoms, Conk-S1 is unusually long (60 amino acids). The only post-translational modification of this peptide is the amidation of the C-terminal carboxylic acid. Conk-S1 shares 33% sequence identity with BPTI and 3...