Importin-�� Is a GDP-to-GTP Exchange Factor of Ran: IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT*

Ran-GTP interacts strongly with importin-β, and this interaction promotes the release of the importin-α-nuclear localization signal cargo from importin-β. Ran-GDP also interacts with importin-β, but this interaction is 4 orders of magnitude weaker than the Ran-GTP·importin-β interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-β promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-β complex stabilizes the complex, which cannot be dissociated by importin-α. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-β has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-β. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-β. Importin-α inhibits the GDP exchange reaction by sequestering importin-β, whereas RanBP1 restores the GDP nucleotide exchange by importin-β by forming a tetrameric complex with importin-β, Ran, and importin-α. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.Ran (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly (1, 2). Like other GTPases, Ran can bind GTP and GDP. Ran-GTP is generated in the nucleus by the guanine exchange factor RCC1 (regulator of chromosome condensation 1), which is associated with the chromatin (3). Ran-GDP is produced in the cytoplasm by the activation of the intrinsic GTPase activity of Ran by RanGAP1 (GTPase-activating protein) (4) and RanBP1 (Ran-binding protein-1, Yrb1p in yeast). The compartmentalization of RanGAP1 (cytoplasm) and RCC1 (nucleus) gives rise to the asymmetric distribution of Ran-GDP (cytoplasm) and Ran-GTP (nucleus) across the nuclear envelope. This asymmetric distribution of Ran-GDP and Ran-GTP plays a central role in nucleocytoplasmic transport by mediating assembly and disassembly of import and export complexes through interaction with the nuclear import machinery (for reviews, see Refs. 5–9).The passage of molecules into the nucleus occurs through the nuclear pore complexes (NPCs)⁶ (10). Nucleocytoplasmic transport is driven by a series of protein-protein interactions and involves several soluble carriers named β-karyopherins. Import carriers are called importins and export carriers are called exportins. The classical nuclear import pathway involves importin-β (Kap95p in yeast) and the adaptor protein importin-α (Kap60p in yeast). In the cytoplasm importin-β binds to importin-α. Their interaction triggers a conformational change of importin-α that increases its affinity for cargo proteins containing a nuclear localization signal (NLS) (11, 12). The translocation of the resulting complex (importin-β·importin-α·NLS) involves interactions with the NPC proteins (nucleoporins), particularly the FXFG-repeat domains (11). The protein cargo is released in the nucleus by the action of Ran-GTP, which induces the dissociation of importin-α from importin-β by forming a stable complex with importin-β. The importins are then recycled to the cytoplasm. Importin-β transfers to the cytoplasm associated with Ran-GTP, and importin-α is exported by CAS (exportin2; Cse1p in yeast) in the form of an importin-α·CAS·Ran-GTP complex (13). Importin-β and importin-α are released from their complexes in the cytoplasm by the combined action of RanBP1 and RanGAP1. Importin-β and importin-α are then able to function in a new cycle of transport, whereas Ran-GDP is transported into the nucleus by NTF2 (nuclear-transport factor-2, Ntf2p in yeast) (14). In the nucleus Ran-GDP is transformed to Ran-GTP by the action of RCC1 (3).The complexity of the nuclear import mechanism is highlighted by the fact that it involves the active participation of soluble factors other than Ran-GTP, importin-β, and importin-α. Indeed, Ran-GDP, RanBP1, and NTF2 have been shown to be involved in the docking and translocation events of nuclear import. Chi et al. (15) have demonstrated that Ran-GDP forms a stable complex with RanBP1 and importin-β; they suggested a role for Ran-GDP in the association of the importin-β·importin-α·NLS complex with the nuclear pore and speculated that the importin-β·importin-α·NLS·Ran-GDP·RanBP1 pentameric complex was the actual translocation complex that moved through the pore. This model has also been adopted by others (16–18) who have proposed that a stable Ran-GDP-containing complex was created on nucleoporin Nup358 (also called RanBP2) and that upon displacement of the importin-β·importin-α·Ran-GDP complex from the RBH (domain homologous to RanBP1) domains of Nup358 by RanBP1, binding of NTF2 triggered translocation to the nucleus. The role of NTF2 as the factor responsible for the translocation of the transport complex through the nuclear envelope has also been proposed by Paschal et al. (19). The role of Ran-GDP and RanBP1 in nuclear import has been demonstrated by a single mutation of a cysteine residue of importin-β; the mutation was required for binding Ran-GDP·RanBP1, but not Ran-GTP·RanBP1, and inhibited the nuclear import in permeabilized cells (20). The active role of RanBP1 in nuclear import has been further demonstrated by Künzler et al. (21), who showed that mutations in the Yrb1 gene encoding the yeast ortholog of RanBP1 impair nucleocytoplasmic transport.Despite considerable evidence for the involvement of Ran-GDP, RanBP1, and NTF2 in nuclear protein import, the precise mechanism by which these molecules regulate this process has been unknown. Here we characterize the interaction between Kap95p and Gsp1p-GDP. We show that this interaction results in GDP-to-GTP exchange on Gsp1p. Furthermore, we demonstrate that Gsp1p, Kap60p, Kap95p, Yrb1p, and Ntf2p interact to regulate the GDP-to-GTP exchange on Gsp1p. We suggest a mechanism of nuclear import additional to the RCC1-dependent pathway that incorporates our observations.     

The Rate-limiting Step in the Cytochrome bc1 Complex (Ubiquinol-Cytochrome c Oxidoreductase) Is Not Changed by Inhibition of Cytochrome b-dependent Deprotonation: IMPLICATIONS FOR THE MECHANISM OF UBIQUINOL OXIDATION AT CENTER P OF THE bc1 COMPLEX*S?

Quinol oxidation at center P of the cytochrome bc₁ complex involves bifurcated electron transfer to the Rieske iron-sulfur protein and cytochrome b. It is unknown whether both electrons are transferred from the same domain close to the Rieske protein, or if an unstable semiquinone anion intermediate diffuses rapidly to the vicinity of the b_L heme. We have determined the pre-steady state rate and activation energy (E_a) for quinol oxidation in purified yeast bc₁ complexes harboring either a Y185F mutation in the Rieske protein, which decreases the redox potential of the FeS cluster, or a E272Q cytochrome b mutation, which eliminates the proton acceptor in cytochrome b. The rate of the bifurcated reaction in the E272Q mutant (<10% of the wild type) was even lower than that of the Y185F enzyme (∼20% of the wild type). However, the E272Q enzyme showed the same E_a (61 kJ mol^-1) with respect to the wild type (62 kJ mol^-1), in contrast with the Y185F mutation, which increased E_a to 73 kJ mol^-1. The rate and E_a of the slow reaction of quinol with oxygen that are observed after cytochrome b is reduced were unaffected by the E272Q substitution, whereas the Y185F mutation modified only its rate. The Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of quinol oxidation (0.7% of the wild type). These results are inconsistent with a sequential “movable semiquinone” mechanism but are consistent with a model in which both electrons are transferred simultaneously from the same domain in center P.The cytochrome bc₁ complex couples the oxidation of a two-electron carrier molecule of quinol to the movement of protons across the inner mitochondrial or bacterial membrane. The key reaction in this energy-conserving mechanism, known as the Q-cycle (1, 2), is the bifurcation of electrons at the active site located closer to the positive side of the membrane, termed center P or Q_o site. One of the electrons from quinol is transferred to a chain of one-electron carriers with relatively high redox potentials that include the FeS cluster of the Rieske protein and the hemes of cytochromes c₁ and c. The other electron is donated to the low potential (b_L) heme of cytochrome b, from which it crosses most of the membrane width to the high potential b_H heme, located close to another active site (center N or Q_i site), where quinone is reduced to quinol after two center P turnovers. Proton release and uptake at each active site are achieved by taking advantage of the chemistry of quinol and quinone, which can only stably exist at physiological pH in the protonated and deprotonated forms, respectively.Critical to the electron bifurcation reaction at center P is the arrangement of protonatable groups (His¹⁸¹ of the Rieske protein and Glu²⁷² of cytochrome b) close to the electron acceptors at opposite sides of the substrate (see Fig. 1). However, the exact mechanism of electron bifurcation at center P is still an unresolved issue. Proposed models have ranged from strictly concerted mechanisms in which both electrons from quinol are extracted simultaneously (3, 4) to those that postulate a highly stabilized semiquinone intermediate (5). Between these two extremes are mechanisms that propose the formation of an unstable semiquinone intermediate after a first electron transfer from quinol to the Rieske protein (6–8), which seem to be supported by recent reports that claim to have detected low concentrations of semiquinone at center P when reoxidation of cytochrome b is impeded under special conditions (9, 10). One version of the unstable semiquinone mechanism proposes that this intermediate diffuses from the vicinity of the Rieske protein to a location within center P located closer to the b_L heme, which would allow non-rate-limiting rates of b_L reduction to occur even at very low semiquinone occupancy (11). In this proposal, the movement of the unstable semiquinone would be allowed by protonation and rotation of Glu²⁷² in cytochrome b, which occupies different conformations in crystallographic structures (Fig. 1) (11–14).Open in a separate windowFIGURE 1.Electron and proton acceptors involved in quinol oxidation at center P. Crystallographic structures 1EZV (12) and 1P84 (13) show stigmatellin (A) or 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole (B) bound at center P forming a hydrogen bond to the His¹⁸¹ residue of the Rieske protein, which is a ligand to the FeS cluster. The Tyr¹⁸⁵ residue in the Rieske protein influences the E_m value of the FeS cluster (22). On the side pointing to the b_L heme, a bound water molecule is also hydrogen-bonded to the inhibitor, either to the Glu²⁷² carboxylate in cytochrome b (A), or to its backbone amino group (B), when the side chain is rotated toward a water network that connects to the propionate of the b_L heme and to Arg⁷⁹ of cytochrome b.An important prediction of the movable semiquinone model (11) is that mutation of Glu²⁷² should impede diffusion of the anionic semiquinone, forcing electron transfer to the b_L heme to occur through a longer distance from the position closer to the Rieske FeS cluster (15), thereby shifting the rate-limiting step from the first to the second electron transfer. Although it has already been reported that different mutations at Glu²⁷² partially slow down quinol oxidation at center P (15–17), no effort has been made so far to evaluate whether the rate-limiting step changes upon inhibition of the deprotonation of quinol (or of a putative semiquinone intermediate) by mutation of the cytochrome b Glu²⁷². In the present work, we analyze the energy of activation of quinol oxidation at center P and show that the rate-limiting step when Glu²⁷² is mutated to glutamine, although slower, is still determined by the driving force for electron transfer to the Rieske protein. We also show that decreasing this driving force enhances the relative inhibition caused by mutating Glu²⁷², suggesting a tight coupling of reactions involved in quinol oxidation and deprotonation. In contrast, reactions with oxygen that bypass the electron bifurcation at center P, which are likely to involve a semiquinone intermediate, are independent of Glu²⁷² and go through an energetic barrier different from that of the bifurcated reaction. We discuss how these results support a mechanism in which both electron transfer events from quinol to the Rieske protein and the b_L heme occur from the same position and at the same time.     

Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: DETERMINANTS OF NUCLEOTIDE SPECIFICITY*S?

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1–3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA)³ as a hexamer and translocates 5′ to 3′ along the DNA strand using the energy of hydrolysis of dTTP (5–7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K_m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in vivo, an equimolar mixture of the full-length gp4 and a truncated form lacking the zinc binding domain of the primase, prefers dTTP and dATP. Therefore, in the present study we have restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7 gene 4 protein, bind nucleotides at the subunit interface (Fig. 1) located between two RecA-like subdomains that bind ATP (11, 12). The location of the nucleotide binding site at the subunit interface provides multiple interactions of residues with the bound NTP. A number of cis- and trans-acting amino acids stabilize the bound nucleotide in the nucleotide binding site and also provide for communication between subunits (13–15). Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is positioned to interact with the γ-phosphate of the bound nucleotide in the adjacent subunit (12, 16). However, His-465 (phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B) interacts with the γ-phosphate of the bound nucleotide in the same subunit (12, 17, 18). The arginine finger and the phosphate sensor have been proposed to couple NTP hydrolysis to DNA unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP hydrolysis (19), and substitution of Asp-424 with Asn leads to a severe reduction in dTTP hydrolysis (20). The conserved Lys-318 in Walker motif A interacts with the β-phosphate of the bound nucleotide and plays an important role in dTTP hydrolysis (21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of the hexameric helicase C-terminal domain of gp4 (17). The structure reveals a ring-shaped molecule with a central core through which ssDNA passes. The inset shows the interface between two subunits of the helicase with adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide binding site. B, the nucleotide binding site of a monomer of the gp4 with the crucial amino acid residues reported earlier and in the present study is shown in sticks. The crystal structures of the T7 gene 4 helicase domain (12) with bound dTTP (C) and dATP (D). The structures shown are the nucleotide binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1 and 1cr2 (12). Arg-504 and Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group of bound dATP. AMPPNP, adenosine 5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are involved in the hydrolysis of dTTP and the coupling of hydrolysis to unwinding, it is intriguing that little information is available on nucleotide specificity. Several crystal structures of T7 helicase in complex with a nucleotide triphosphate are available. However, most of structures were crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was diffused into the crystal. The crystal structure of the T7 helicase domain bound with dTTP or dATP was reported by Sawaya et al. (12). These structures assisted us in identifying two basic residues (Arg-363 and Arg-504) in close proximity to the sugar and base of the bound nucleotide whose orientation suggested that these residues could be involved in nucleotide selection. Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the subunit interface of the hexameric helicase (Fig. 1). Arg-504 and Tyr-535 are structurally well conserved in various helicases (12). However, Arg-504 could make a hydrogen bridge with the OH group of thymidine, thus suggesting a role in dTTP specificity. On the other hand, Arg-363 is in close proximity (∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the dTTP-bound structure this residue is displaced by 7.12 Å (Fig. 1) from the equivalent position. Consequently Arg-363 could play a role in dATP binding. The crystal structures do not provide any information on different interaction of residues with the phosphates of dATP and dTTP. However, alignment of the residues in the P-loops of different hexameric helicases reveals that the serine adjacent to the invariant lysine at position 319 (Ser-319) is conserved in bacteriophages, whereas bacterial helicases have a conserved threonine in the equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and bacteriophage T4 gene 41 uses rGTP or rATP (22).Although considerable information is available on the role of residues in nucleotide binding and dTTP hydrolysis, very little is known on the determinants of nucleotide specificity. In the present study we made an attempt to address the role of a few selected residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both of which are hydrolyzed and mediate DNA unwinding. We show that under physiological conditions T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.     

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells: IMPLICATIONS FOR AGGREGATION AND CELLULAR PROPERTIES OF α-SYNUCLEIN

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.     

Two Snapshots of Electron Transport across the Membrane: INSIGHTS INTO THE STRUCTURE AND FUNCTION OF DsbD*S?

In Escherichia coli, the periplasmic protein disulfide isomerase, DsbC, is maintained reduced by transfer of electrons from cytoplasmic thioredoxin-1 (Trx1) via the cytoplasmic membrane protein, DsbD. The transmembrane domain of DsbD (DsbDβ), which comprises eight transmembrane segments (TMs), contains two redox-active cysteines (Cys-163 and Cys-285), each of which is water-exposed to both sides of the membrane. Cys-163 in TM1 and Cys-285 in TM4 can interact with cytoplasmic Trx1 and a periplasmic Trx-like domain of DsbD, respectively. When Cys-163 and Cys-285 are disulfide-bonded, the C-terminal halves of TM1 and TM4 are water-exposed, whereas the N-terminal halves of these TMs are not. To assess possible conformational changes of DsbDβ when its two cysteines are reduced, we have determined the accessibility of portions of TM1 and TM4. We substituted cysteines for amino acids in these TM segments and determined alkylation accessibility. We find that the alkylation accessibility of single Cys replacements in TM1 and TM4 is the same in oxidized and reduced DsbDβ, indicating a relatively static conformation of DsbDβ between the two redox states. We also find that the accessibility of amino acids of TM2 and TM3 when Cys-163 and Cys-285 are oxidized or reduced shows no change. Together, these results support a relatively static structure of DsbDβ in the switch between the oxidized and the reduced state but raise the possibility of conformational changes when interacting with Trx proteins. In addition, we also find water-exposed residues in the cytoplasmic proximal portion of TM3, allowing a more detailed characterization of the cavity in DsbDβ.The cell envelope of most bacteria is an oxidizing environment. In many bacteria, the main oxidant system consists of DsbA and DsbB. DsbA introduces disulfide bonds into newly synthesized and secreted polypeptides containing cysteines and is regenerated as an oxidative enzyme by the membrane protein DsbB. Electrons are ultimately transferred from DsbB to the respiratory chain (1–3). However, there are also certain cell envelope proteins that require a reductive enzyme to act on them. This is the case for those proteins that contain multiple cysteines and that are often misoxidized by DsbA, thus generating non-native disulfide bonds. The protein DsbC, a protein disulfide isomerase, can promote rearrangement of such incorrect disulfide bonds, resulting in a correctly folded protein (4–7). It does this either by using the reduced cysteine in its active site to resolve non-native disulfide bonds and promoting the formation of the native pairs or simply by reducing the substrate protein, which may be correctly oxidized by DsbA and given a second chance (8). In the latter mechanism, DsbC becomes oxidized and must be reduced. This reduction is carried out by a cytoplasmic membrane protein, DsbD, which receives electrons for this purpose from thioredoxin-1 (Trx1)² in the cytoplasm (5, 9).DsbD is composed of three domains, each containing two redox-active cysteines (Fig. 1). DsbDβ, the membrane-embedded domain containing eight transmembrane segments (TMs), receives electrons from Trx1 and then transfers them to the C-terminal periplasmic domain, DsbDγ, which contains a Trx-like fold (10–15). The N-terminal periplasmic domain, DsbDα, which contains an immunoglobulin-like fold, is then reduced by DsbDγ and transfers electrons to DsbC (13, 16, 17).Open in a separate windowFIGURE 1.Electron transfer pathway through transmembrane domain (β) of DsbD and its membrane topology predicted from the primary sequence. The topology of DsbDβ was predicted using HMMTOP. The essential two cysteines are shown in bold without a circle and numbered. The residues indicated with a star in TM1 and TM4 are water-exposed when the Cys-163 and Cys-285 are disulfide-bonded (19). Studies on the residues in TM2 and TM3 are shown in Fig. 4. The essential cysteines in the other domains (α and γ) and interacting proteins (Trx1 and DsbC) are shown in a white S (the sulfur of thiol) in gray circles. The tailless arrows indicate where a signal sequence of DsbD and three hemagglutinin (HA) epitopes are fused at the N terminus of DsbDβ, and a c-Myc epitope is fused at the C terminus of DsbDβ. The figure in the inset describes the model of DsbDβ, in which Cys-163 and Cys-285 form a disulfide bond in the middle of the protein and halves of C-terminal TM1 and TM4 are water-exposed (black; C-TM1 and C-TM4), whereas those of N-terminal ones are not (N-TM1 and N-TM4).Electron transfer through the transmembrane domain DsbDβ is quite unusual when compared with that of other membrane electron transport proteins. Required extrinsic factors, for example, quinone, FAD, heme, or metal centers, which are often used as cofactors for electron transfer, have not been found (18). As a result, it is proposed that thiol-disulfide exchange reactions alone promote the transfer of electrons across the cytoplasmic membrane, utilizing the two cysteines, Cys-163 and Cys-285 of DsbDβ. Evidence for this mechanism comes from the detection of likely reaction intermediates including the Cys-163–Cys-285 disulfide and mixed disulfide complexes, Trx1-DsbDβ(Cys-163) and DsbDβ(Cys-285)-DsbDγ (13, 14, 19).We have previously studied the accessibility to the aqueous environment of amino acids in TM1 and TM4 of DsbDβ, which contain Cys-163 and Cys-285, respectively. Our results, in conjunction with a comparison of the amino acid sequences of TM1–3 and TM4–6 of DsbDβ, suggest antiparallel and pseudosymmetrical properties of TM1 and TM4 (19, 20). Cys-163 in TM1 and Cys-285 in TM4 are water-exposed to both sides of the membrane when they are in the reduced state and suggested to be located in the middle of the membrane (helices). When Cys-163 and Cys-285 are disulfide-bonded, the proximal portion of the cytoplasmic side of TM1 at the C terminus of Cys-163 and that of the periplasmic side of TM4 at the C terminus of Cys-285 are highly water-exposed, whereas the other portion of each TM is not. Therefore, we proposed an hourglass-like model (Fig. 1, inset) and suggested that the water-exposed halves of TM1 and TM4 are cavity-located non-membrane-spanning helices and involved in the interactions of Trx1 and DsbDγ, respectively.However, in our previous studies (19), we did not determine whether, when DsbDβ is in the reduced state, the arrangement of TM1 and TM4 or other TMs would be similar to that seen when Cys-163 and Cys-285 are disulfide-bonded. Doing this comparison is important because it has been proposed that alternative exposure of the cysteines to the aqueous environment depending on their redox states explains the electron transfer process across the membrane (Refs. 21–23; see “Discussion”). In addition, to further define the structural features of DsbDβ, we wished to determine how TM segments other than TM1 and TM4 are arranged in terms of their water accessibility. In this study, we examined the accessibility of many residues in TM1 and TM4, as well as studying the arrangements of TM2 and TM3, using site-directed cysteine alkylation in both redox states.Our data show that the four TMs studied, TM1, TM2, TM3, and TM4, have similar accessibility properties whether DsbDβ is in the oxidized or reduced state. We also find additional water-exposed residues in the proximal portion of the cytoplasmic side of TM3.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

Structural Evidence of Substrate Specificity in Mammalian Peroxidases: STRUCTURE OF THE THIOCYANATE COMPLEX WITH LACTOPEROXIDASE AND ITS INTERACTIONS AT 2.4 ? RESOLUTION*S?

The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN^–) has been determined at 2.4Å resolution. It revealed that the SCN^– ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN^– ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN^– forms a hydrogen bond with a water (Wat) molecule at position 6′. This water molecule is stabilized by two hydrogen bonds with Gln⁴²³ N^ε2 and Phe⁴²² oxygen. In contrast, the placement of the SCN^– ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln⁴²³, Phe⁴²² oxygen, and Wat⁶′ in LPO is occupied primarily by the side chain of Phe⁴⁰⁷ in MPO due to an entirely different conformation of the loop corresponding to the segment Arg⁴¹⁸–Phe⁴³¹ of LPO. This arrangement in MPO does not favor a similar orientation of the SCN^– ion. The orientation of the catalytic product OSCN^– as reported in the structure of LPO·OSCN^– is similar to the orientation of SCN^– in the structure of LPO·SCN^–. Similarly, in the structure of LPO·SCN^–·CN^–, in which CN^– binds at Wat¹, the position and orientation of the SCN^– ion are also identical to that observed in the structure of LPO·SCN.Lactoperoxidase (LPO⁴; EC 1.11.1.7) is a Fe³⁺ heme enzyme that belongs to the mammalian peroxidase family (1). The family of mammalian peroxidases comprises lactoperoxidase (2), eosinophil peroxidase (3), thyroid peroxidase (4), and myeloperoxidase (MPO) (5). LPO, eosinophil peroxidase, and MPO are responsible for antimicrobial function and innate immune responses (6–8), whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis (9). These peroxidases are different from plant and fungal peroxidases because unlike plant and fungal enzymes, the prosthetic heme group in mammalian peroxidases is covalently linked to the protein (10). There are also several striking structural and functional differences among the mammalian peroxidases (11). The heme group in MPO is attached to the protein via three covalent linkages (12), whereas LPO (12, 13), eosinophil peroxidase (12), and thyroid peroxidase (12) contain only two ester linkages. These covalent and various non-covalent linkages contribute differentially to the high stability of the heme core as well as for the peculiar values of their redox potentials (2, 14). Furthermore, MPO consists of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase, and thyroid peroxidase are single chain proteins, although their chain lengths differ greatly. In addition, their sequences contain several critical amino acid differences that may also contribute to the variations in the stereochemical environments of the substrate-binding sites. As a consequence of these differences, the mammalian enzymes oxidize various inorganic ions such as SCN^–, Br^–, Cl^–, and I^– with differing specificities and potencies. Biochemical studies have shown that LPO catalyzes preferentially the conversion of SCN^– to OSCN^– (15, 16), whereas MPO uses halides (17, 18) with a preference for chloride ion as the substrate. The preferences of eosinophil peroxidase and thyroid peroxidase are bromide and iodide, respectively. However, the stereochemical basis of the reported preferences for the substrates by mammalian heme peroxidases is still unclear. So far, the structures of only two mammalian enzymes, MPO and LPO, have been determined (12, 13). It is of considerable importance to identify the structural parameters that are responsible for the subtle specificities. In the present work, we have attempted to address this question through the new crystal structures of LPO complexes with SCN^– ions using goat, bovine, and buffalo lactoperoxidases. Because the overall structures of complexes of SCN^– with LPO from all three species were found to be identical, the structure of the complex of buffalo LPO with SCN^– and the ternary complex with SCN^– and CN^– will be discussed here, and buffalo LPO will be termed hereafter as LPO. To highlight the factors pertaining to binding specificity of SCN^–, a comparison of the structures of LPO·SCN^– and MPO·SCN^– has also been made, revealing many valuable differences pertaining to the observed orientations of the common substrate, SCN^– ion, when bound at the substrate-binding site in the distal heme cavity of the two structures. The structures of LPO·SCN^– and MPO·SCN^– clearly show that the bound SCN^– ions are present in the distal heme cavity of two enzymes with opposite orientations. In the structure of LPO·SCN^–, the sulfur atom is closer to the heme iron than the nitrogen atom, whereas in that of MPO·SCN^–, the nitrogen atom is closer to the heme iron than the sulfur atom. As a result of this, the interactions of the SCN^– ion in the distal site of two proteins differ drastically. Gln⁴²³, a conserved water (Wat) molecule at position 6′, and a well aligned carbonyl oxygen of Phe⁴²² in the proximity of the substrate-binding site in LPO against a protruding Phe⁴⁰⁷ in MPO seem to play the key roles in inducing the observed orientations of SCN^– ions in LPO and MPO. The structure of LPO·SCN^– has also been compared with the structure of its ternary complex with SCN^– and CN^– ions.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.     

THE ETHICS OF SHAM SURGERY IN PARKINSON'S DISEASE: BACK TO THE FUTURE?

Despite intense academic debate in the recent past over the use of ‘sham surgery’ control groups in research, there has been a recent resurgence in their use in the field of neurodegenerative disease. Yet the primacy of ethical arguments in favour of sham surgery controls is not yet established. Preliminary empirical research shows an asymmetry between the views of neurosurgical researchers and patients on the subject, while different ethical guidelines and regulations support conflicting interpretations. Research ethics committees faced with a proposal involving sham surgery should be aware of its ethical complexities. An overview of recent and current placebo‐controlled surgical trials in the field of Parkinson's Disease is provided here, followed by an analysis of the key ethical issues which such trials raise.