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...

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

The high affinity of human plasma β2-glycoprotein I (β 2 GPI), also known as apolipoprotein-H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. β 2 GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the β 2 GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central β-spiral of four antiparallel β-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313-316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The β 2 GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of β 2 GPI.

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices ␣5 and ␣6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.Members of the Bcl-2 protein family are essential players in the complex regulation of apoptosis (1, 2). They are divided into three subgroups: the anti-apoptotic Bcl-2-like proteins, the pro-apoptotic multidomain proteins (Bax and Bak), and the pro-apoptotic BH3 3 -only proteins. To keep programmed cell death under control, Bax activation needs to be strictly regulated, as abnormal cell death is disadvantageous for multicellular organisms.Following cleavage by caspase-8, the BH3-only protein, Bid, is known to activate Bax (3-6). Recently the events involved in BaxBid interaction were investigated by fluorescent techniques (7). Bax is activated through a cascade of conformational changes from being inactive and cytosolic to an oligomeric, membrane-inserted state. In the mitochondrial outer membrane (8, 9) activated Bax is responsible for cytochrome c release and apoptosis initiation (10). Bax oligomerization has been shown to occur also in vitro by incubation with detergents (10 -14).The structures of monomeric Bax and Bid were solved by NMR (14 -16). Bax has a globular fold composed of nine ␣-helices (␣1 to ␣9), with ␣2 representing the BH3 domain and ␣5/␣6 the hydrophobic hairpin (see Fig. 1A). Similarities in structure and function with the monomeric inactive form of the channel-forming domain of bacterial colicins or diphtheria toxins are evident (14, 17). The BH3-only protein, Bid, shows a similar globular fold but lacks ␣8 and ␣9 and has an additional short helix (␣1/2) between ␣1 and ␣2. The structure of active Bax is still unknown, but helices ␣5, ␣6, and ␣9 are reported to in...

P II proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P II and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P II target, whose activity responds to 2-OG via P II . Here we show structures of the Synechococcus elongatus P II protein in complex with ATP, Mg 2þ , and 2-OG, which clarify how 2-OG affects P II -NAGK interaction. P II trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P II trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg 2þ ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B-and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P II target. Structures of P II trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P II senses effectors in a wide range of concentrations.metabolic signaling | nitrogen regulation | cyanobacteria | chloroplasts T he P II proteins constitute one of the largest and most widely distributed family of signal transduction proteins present in archaea, bacteria, and plants. They control key processes of nitrogen metabolism in response to central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance (1-4). These effectors bind to P II in an interdependent manner (see below), thereby transmitting metabolic information into structural states of this sensor protein (3, 5). Furthermore, P II proteins may be posttranslationally modified (1, 6). Depending on the signal input states, P II proteins bind and thereby regulate the activity of key metabolic and regulatory enzymes, transcription factors, or transport proteins (1-3). In cyanobacteria and plants, the controlling enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase (NAGK), is a major P II target (7-9). Moreover, P II affects gene expression in cyanobacteria through binding to the transcriptional coactivator of NtcA, PipX (10). In plants, acetyl-CoA carboxylase was recently shown to be regulated by P II , providing an additional link between carbon and nitrogen regulation (11). Although these P II targets share no common structural element, interaction with P II is inhibited by 2-OG.P II proteins are homotrimers of 12-to 13-kDa subunits, built of a double ferredoxin-like fold-containing core (βαβ-βαβ), with a characteristic and highly cons...

The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to the OB rings.

Members of the AAA+ superfamily have been identi¢ed in all organisms studied to date. They are involved in a wide range of cellular events. In bacteria, representatives of this superfamily are involved in functions as diverse as transcription and protein degradation and play an important role in the protein quality control network. Often they employ a common mechanism to mediate an ATP-dependent unfolding/disassembly of protein^protein or DNA^protein complexes. In an increasing number of examples it appears that the activities of these AAA+ proteins may be modulated by a group of otherwise unrelated proteins, called adaptor proteins. These usually small proteins speci¢cally modify the substrate recognition of their AAA+ partner protein. The occurrence of such adaptor proteins are widespread; representatives have been identi¢ed not only in Escherichia coli but also in Bacillus subtilis, not to mention yeast and other eukaryotic organisms. Interestingly, from the currently known examples, it appears that the N domain of AAA+ proteins (the most divergent region of the protein within the family) provides a common platform for the recognition of these diverse adaptor proteins. Finally, the use of adaptor proteins to modulate AAA+ activity is, in some cases, an elegant way to redirect the activity of an AAA+ protein towards a particular substrate without necessarily a¡ecting other activities of that AAA+ protein while, in other cases, the adaptor protein triggers a complete switch in AAA+ activity. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These hostdefense 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.crystallography | electrophysiology | ion conduction | molecular dynamics