TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the Cterminal nudix domain. We also show that oxidative/ nitrosative stress-induced gating can be inhibited by pharmacological reagents predicted to inhibit NAD hydrolysis to ADP-ribose and by suppression of ADP-ribose accumulation by cytosolic or mitochondrial overexpression of an enzyme that specifically hydrolyzes ADP-ribose. Overall, our data are most consistent with a model of oxidative and nitrosative stress-induced TRPM2 activation in which mitochondria are induced to produce free ADP-ribose and release it to the cytosol, where its subsequent accumulation induces TRPM2 gating via interaction within a binding cleft in the C-terminal NUDT9-H domain of TRPM2.Eukaryotic cells have been shown to react and adapt to conditions of oxidative stress produced by disulfide-inducing chemicals, reactive oxygen species (ROS), 1 or reactive nitrogen species (RNS) through a variety of redox-sensitive signaling pathways (variously reviewed in Refs. 1-12). Recently, the TRPM2 cation channel has been shown to undergo gating in response to oxidative or nitrosative stress occurring upon ROS or RNS exposure (13,14). As TRPM2 is a novel dual function protein that possesses both ion channel and ADP-ribose hydrolase functions (15, 16), there has been great interest in understanding the molecular mechanisms through which oxidative and nitrosative stress activate TRPM2 channels, as well as the physiological role(s) of TRPM2-mediated ion fluxes and enzymatic activity in the context of cellular exposure to ROS and RNS.TRPM2 is classified as a member of the transient receptor potential melastatin-related (TRPM) ion channel family based on the homology of its Ϸ600 amino acid N-terminal domain and Ϸ300 amino acid channel-forming domain to corresponding regions of other TRPM family members. The enzymatic function of TRPM is encoded by a C-terminal domain, designated the NUDT9-homology (NUDT9-H) domain, which is homologous to the NUDT9 ADP-ribose hydrolase (15,17). In vitro studies of TRPM2 channel function using patch clamp techniques have suggested that ADP-ribose and NAD are both able to directly induce gating of full-length TRPM2 channels (14,15,18), and in vitro studies of the NUDT9-H domain demonstrate that it has a low level of ADP-ribose hydrolase activity and the apparent capacity to interact with NAD through its nudix hydrolase enzymatic motif (14, 15). However, there is a significant gap in understanding how the in vitro data on TRPM2 function relate to oxidative/nitrosative stress-induced TRPM2 gating in intact c...

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.

Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 do IntroductionThe principal mechanism used to stop the loss of blood in mammals following vascular injury consists of a pair of overlapping proteolytic cascades called the extrinsic and intrinsic pathways. [1][2][3][4] The process of blood coagulation requires extraordinary spatial and temporal regulation, which is accomplished by assembling and tethering the central proteolytic activities of these cascades at the location of transiently exposed biomolecules and cellular surfaces ( Figure 1A). This includes an integral membrane protein called "tissue factor" that initiates the rapid up-regulation of the short-lived extrinsic pathway, 5 and the surfaces of activated platelets, which modulate the activation of the longer-lived intrinsic pathway. 6 A total of 2 homologous procoagulants, factors V and VIII (fV and fVIII), are each localized on the surface of these platelets, where they nucleate the assembly of multiprotein proteolytic complexes.When fVIII is bound to activated platelets at the site of vascular injury, it recruits the serine protease fIXa into a complex that then catalyzes the proteolytic activation of fX. 1,4,7 The proteolytic activity of fIXa is enhanced by approximately 200 000-fold through its interaction with fVIII, calcium, and the phospholipid bilayer, 8 corresponding to an increase of approximately 10 9 in k cat /K M .The full-length, unprocessed fVIII protein consists of 2332 amino acid residues and has the domain structure A1-A2-B-A3-C1-C2 9-12 ( Figure 1B). The 3 A domains are each approximately 330 residues, and approximately 40% identical to each other and to the copper-binding protein ceruloplasmin. 13 The C domains are smaller (approximately 160 residues) and are more distantly related to various members of the discoidin protein fold family, such as galactose oxidase. [14][15][16][17] The B domain has no known structural homologs, is heavily glycosylated, and is relatively dispensible for procoagulant activity. fVIII is initially processed by proteolytic cleavage events that remove a large portion of the B domain, generating a heterodimer that circulates in a tight complex with von Willebrand factor (VWF). 18 This interaction is essential for maintaining stable levels of fVIII in circulation. 19 Upon vascular injury, further proteolytic processing generates activated factor VIIIa (fVIIIa), a heterotrimer (A1/A2/A3-C1-C2) that is released from VWF and binds to activated platelets. 18 The carboxy-terminal 159 amino acids of fVIII comprise its C2 domain, which is invo...

We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the ␣-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% ␣-helical content. In contrast, the ␣-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in ␣-helicity. Interaction of a 90-residue NH 2 -terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in ␣-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.Neurotransmitters are released from presynaptic nerve endings by Ca 2ϩ -triggered exocytosis of synaptic vesicles. Several lines of evidence suggest that exocytotic membrane fusion is mediated by a complex of conserved proteins which includes the synaptic vesicle protein synaptobrevin (also referred to as vesicle-associated membrane protein or VAMP) and the synaptic membrane proteins syntaxin and SNAP-25. Homologs of these neuronal proteins have been identified in many nonneuronal cell types including the yeast Saccharomyces cerevisiae, suggesting that the mechanism of exocytotic membrane fusion is conserved in all eukaryotic cells (for review, see Refs. 1-4).Although the evidence linking these proteins to membrane fusion is quite compelling, very little is known about their mechanism of action. Rothman and colleagues (5) found that the three membrane proteins form a complex that interacts with additional soluble proteins known to support membrane fusion in cell-free extracts (5). These soluble proteins include the SNAPs 1 (soluble NSF attachment proteins) with three isoforms (␣-, ␤-(brain-specific), and ␥-SNAP), and the ATPase NSF (N-ethylmaleimide-sensitive fusion protein). SNAPs and NSF apparently operate on all relatives of the synaptobrevin/ syntaxin/SNAP-25 protein families, which are therefore commonly referred to as SNAREs (SNAP receptors). ATP hydrolysis by NSF leads to disassembly of the synaptobrevin-syntaxin-SNAP-25-complex (6), an effect that is associated with a different state of syntaxin (7,8). Ultimately, vesicle docking and membrane fusion can be viewed as a series of sequential protein assembly and disassembly steps which may involve structural changes and regulated interactions of at least some of the proteins with the participating phospholipid bilayers. I...

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Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F- actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

COMPOSITIONAL ANALYSIS The basic compositional data used in our analysis were taken from a recent critical compilation by Scanu and Kruski (6). The reported data are based on dry weight and thus they do not take into consideration the possible presence of water in the lipoprotein particle. In our analysis we also assumed the absence of significant amounts of structural water inside lipoproteins. Minor components, such as free fatty acid, carbohydrates, vitamins, and unidentified compounds, were ignored because, at best, they amount to not more than 5% of the total weight (6) and are likely not to play an essential structural role. For very low density lipoprotein (VLDL) and chylomicrons Scanu and Kruski presented only average compositional data. VLDL and chylomicrons are, in fact, particles of rather broad size distribution, and several authors reported compositional data on their subfractions (2, 7). These additional data on subfractions were also used wherever appropriate. Table 1 summarizes the weight percentage compositions of the major constituents of all lipoprotein classes.From the molecular weight and the weight percentage composition, assuming no structural water, one can calculate the number of molecules of each component in a lipoprotein particle ( Table 2). The following molecular weights were used for the calculations: triglycerides, 850; phospholipid, 775; cholesterol ester, 650; and cholesterol, 387. For the protein content it was not useful to calculate the number of protein molecules per particle because the various lipoprotein classes contain a variety of apoproteins of different molecular weights. For comparison it was more suitable to calculate the number of amino acid residues per particle, assuming an average residue weight of 100. Assuming that all lipoproteins are spherical particles, one can calculate from the molecular weight and the density of the particle the radius of an equivalent sphere r in units of A, also shown in Table 2. Since for the average VLDL and chylomicrons no reliable molecular 'weight data were available, we calculated the molecular weight from the experimentally determined average particle radii, 200 A and 600 A, respectively. Triglyceride and cholesterol ester compositionThe major components of lipoproteins can be divided into nonpolar lipids (triglycerides and cholesterol esters) and polar constituents (phospholipids, cholesterol, and protein). If hydrophobic interactions play an important part in organizing the components, then one would expect the existence of a hydrophobic phase, as it has already been suggested for VLDL and 837