The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a ␤-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 Å resolution, revealing a high-resolution image of its architecture formed by 19 ␤-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltagesensing N-terminal segment is clearly resolved. The ␣-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.␤-barrel ͉ mitochondria ͉ outer membrane protein ͉ ATP flux A ll eukaryotic cells require efficient exchange of metabolites between the cytoplasm and the mitochondria. This exchange is mediated by the most abundant protein in the outer mitochondrial membrane, the voltage-dependent anion channel (VDAC), which facilitates movement of ions and metabolites between the cytoplasm and the intermembrane space of the mitochondria. VDAC was first discovered in 1976 (1) and has since been extensively studied by a number of biochemical and biophysical techniques demonstrating its conserved properties of voltage gating and ion selectivity and its ability to act as a scaffold for modulator proteins from both sides of the outer membrane (2, 3).Single-channel conductance experiments on VDAC1 at low membrane potential (10 mV) show a high conductance indicative of a large pore, often referred to as the open state of the channel (2). As voltage is increased (Ͼ30 mV) in either a positive or negative direction, a lower conductance, ostensibly the closed state, is obtained. Endogenous potentials caused by chemical gradients across the outer membrane [Donnan potentials (4)] may thus be sufficient to regulate this channel. Although the nature of either of these states is unknown in the absence of structural data, transition between them presumably involves conformational changes constituting a gating action that hinders the passage of metabolites such as adenine nucleotides. Furthermore, this transition is associated with altered ion selectivity because the channel shifts from weakly anion selective to weakly cation selective as it moves from open to closed. This complex gating behavior has driven numerous investigations that have provided sometimes contradictory findings; however, the role of VDAC to regulate metabolite traffic across the outer membrane is firmly established.As the major pathway into and out of mitochondria, VDAC mediates an intimate dichotomy between metabolism and death in all cells (5). Mitochondrial-dependent cell death involves numerous proteins [including hexokinase (6) and the Bcl-2 family of proteins (7), in particular] that alternatively promote or prevent mitochondrial dysfunction through interaction with, and potentially m...

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ~3.0Å structure contains 14 transmembrane helices in an inward facing conformation with a core structure of inverted repeats of 5 TM helices (TM2-TM6 and TM7-TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.

Members of the fibroblast growth factor (FGF) family of proteins stimulate the proliferation and differentiation of a variety of cell types through receptor-mediated pathways. The three-dimensional structures of two members of this family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. The locations of sequences implicated in receptor and heparin binding by FGF are presented. These sites include beta-sheet strand 10, which is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family, and which shows sequence conservation among the FGF family and interleukin-1 beta.

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn2+, which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.

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Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

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