Extended structure of rat islet amyloid polypeptide in solution.

The process of islet amyloid polypeptide (IAPP) formation and the prefibrillar oligomers are supposed to be one of the pathogenic agents causing pancreatic ß-cell dysfunction. The human IAPP (hIAPP) aggregates easily and therefore, it is difficult to characterize its structural features by standard biophysical tools. The rat version of IAPP (rIAPP) that differs by six amino acids when compared with hIAPP, is not prone to aggregation and does not form amyloid fibrils. Similar to hIAPP it also demonstrates random-coiled nature in solution. The structural propensity of rIAPP has been studied as a hIAPP mimic in recent works. However, the overall shape of it in solution still remains elusive. Using small angle X-ray scattering (SAXS) measurements combined with nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) the solution structure of rIAPP was studied. An unambiguously extended structural model with a radius of gyration of 1.83 nm was determined from SAXS data. Consistent with previous studies, an overall random-coiled feature with residual helical propensity in the N-terminus was confirmed. Combined efforts are necessary to unambiguously resolve the structural features of intrinsic disordered proteins.

Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207.

The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε(103-120)). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ß subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F(1)F(O) ATP synthase in M. tuberculosis.

The stem region of premembrane protein plays an important role in the virus surface protein rearrangement during dengue maturation.

BACKGROUND: Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition. RESULTS: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic. CONCLUSION: At acidic condition, E proteins are attracted to the membrane-associated prM-stem. SIGNIFICANCE: prM-stem region induces virus structural changes during maturation. Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111-131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.

Relevance of the conserved histidine and asparagine residues in the phosphate-binding loop of the nucleotide binding subunit B of A1A0 ATP synthases.

The nucleotide binding sites in A-ATP synthases are located at the interfaces of subunit A and B, which is proposed to play a regulatory role. Differential binding of MgATP and -ADP to subunit B has been described, which does not exist in the related α and B subunits of F-ATP synthases and V-ATPases, respectively. The conserved phosphate loop residues, histidine and asparagine, of the A-ATP synthase subunit B have been proposed to be essential for γ-phosphate interaction. To investigate the role of these conserved P-loop residues in nucleotide-binding, subunit B residues H156 and N157 of the Methanosarcina mazei Gö1 A-ATP synthase were separately substituted with alanine. In addition, N157 was mutated to threonine, because it is the corresponding amino acid in the P-loop of F-ATP synthase subunit α. The structures of the subunit B mutants H156A, N157A/T were solved up to a resolution of 1.75 and 1.7 Å. The binding constants for MgATP and -ADP were determined, demonstrating that the H156A and N157A mutants have a preference to the nucleotide over the wild type and N157T proteins. Importantly, the ability to distinguish MgATP or -ADP was lost, demonstrating that the histidine and asparagine residues are crucial for nucleotide differentiation in subunit B. The structures reveal that the enhanced binding of the alanine mutants is attributed to the increased accessibility of the nucleotide binding cavity, explaining that the structural arrangement of the conserved H156 and N157 define the nucleotide-binding characteristics of the regulatory subunit B of A-ATP synthases.

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk.

A(1)A(O) ATP synthases are the major energy converters of archaea. They are composed of an A(1) region that synthesizes ATP and an integral part A(O) that conducts ions. Subunit E is a component of the peripheral stalk that links the A(1) with the A(O) part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases.

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A(1)A(O) ATP synthase of Methanosarcina mazei Gö1.

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A(1)A(O) ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A(1)A(O) ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.

Binding of subunit E into the A-B interface of the A(1)A(O) ATP synthase.

Two of the distinct diversities of the engines A(1)A(O) ATP synthase and F(1)F(O) ATP synthase are the existence of two peripheral stalks and the 24kDa stalk subunit E inside the A(1)A(O) ATP synthase. Crystallographic structures of subunit E have been determined recently, but the epitope(s) and the strength to which this subunit does bind in the enzyme complex are still a puzzle. Using the recombinant A(3)B(3)D complex and the major subunits A and B of the methanogenic A(1)A(O) ATP synthase in combination with fluorescence correlation spectroscopy (FCS) we demonstrate, that the stalk subunit E does bind to the catalytic headpiece formed by the A(3)B(3) hexamer with an affinity (K(d)) of 6.1±0.2µM. FCS experiments with single A and B, respectively, demonstrated unequivocally that subunit E binds stronger to subunit B (K(d)=18.9±3.7µM) than to the catalytic A subunit (K(d)=53.1±4.4). Based on the crystallographic structures of the three subunits A, B and E available, the arrangement of the peripheral stalk subunit E in the A-B interface has been modeled, shining light into the A-B-E assembly of this enzyme.

Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% ß-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.

Crystal and solution structure of the C-terminal part of the Methanocaldococcus jannaschii A1AO ATP synthase subunit E revealed by X-ray diffraction and small-angle X-ray scattering.

The structure of the C-terminus of subunit E (E(101-206)) of Methanocaldococcus jannaschii A-ATP synthase was determined at 4.1 A. E(101-206) consist of a N-terminal globular domain with three alpha-helices and four antiparallel beta-strands and an alpha-helix at the very C-terminus. Comparison of M. jannaschii E(101-206) with the C-terminus E(81-198) subunit E from Pyrococcus horikoshii OT3 revealed that the kink in the C-terminal alpha-helix of E(81-198), involved in dimer formation, is absent in M. jannaschii E(101-206). Whereas a major dimeric surface interface is present between the P. horikoshii E(81-198) molecules in the asymmetric unit, no such interaction could be found in the M. jannaschii E(101-206) molecules. To verify the oligomeric behaviour, the low resolution structure of the recombinant E(85-206) from M. jannaschii was determined using small angle X-ray scattering. Rigid body modeling of two copies of one of the monomer established a fit with a tail to tail arrangement.

Purification and crystallization of the entire recombinant subunit E of the energy producer A(1)A(o) ATP synthase.

A(1)A(o) ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3 A resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25 A, and contained three molecules in the asymmetric unit.

Structural determination of functional units of the nucleotide binding domain (NBD94) of the reticulocyte binding protein Py235 of Plasmodium yoelii.

BACKGROUND: Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH. METHODOLOGY/PRINCIPAL FINDINGS: In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments. CONCLUSIONS: These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

The effect of NBD-Cl in nucleotide-binding of the major subunit alpha and B of the motor proteins F1FO ATP synthase and A1AO ATP synthase.

Subunit alpha of the Escherichia coli F(1)F(O) ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of alpha allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K ( d )) of 1.6 microM of bound MgATP-ATTO-647N and 2.9 microM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 microM and 55 microM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC(50)), respectively. In contrast, no effect was observed in the presence of N,N'-dicyclohexylcarbodiimide. As subunit alpha is the homologue of subunit B of the A(1)A(O) ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC(50) values of 41 microM and 42 microM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.

The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains.

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains.

Association of the eukaryotic V1VO ATPase subunits a with d and d with A.

Owing to the complex nature of V(1)V(O) ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V(1) headpiece and the V(O)-domain of the yeast V(1)V(O) ATPase via subunit A and d as well as the V(O) subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A(3)B(3) hexamer with V(O).

Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A1Ao ATP synthase.

A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine-diphosphate binding to subunit B of the A(1)A(O) adenosine triphosphate (ATP) synthase from Methanosarcina mazei Gö1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X-ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 A resolution, showing the substituted Trp-residue inside the predicted adenine-binding pocket. The cocrystallized ATP molecule could be trapped in a so-called transition nucleotide-binding state. The high resolution structure shows the phosphate residues of the ATP near the P-loop region (S150-E158) and its adenine ring forms pi-pi interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F(1)F(O) ATP synthases, is discussed in light of a transition nucleotide-binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A(3)B(3)- and A(3)B(R416W)(3)-subcomplex, composed of subunit A and the B subunit mutant R416W, of the A(1)A(O) ATP synthase is shown.

ATP/ADP binding to a novel nucleotide binding domain of the reticulocyte-binding protein Py235 of Plasmodium yoelii.

The mechanism by which a malaria merozoite recognizes a suitable host cell is mediated by a cascade of receptor-ligand interactions. In addition to the availability of the appropriate receptors, intracellular ATP plays an important role in determining whether erythrocytes are suitable for merozoite invasion. Recent work has shown that ATP secreted from erythrocytes signals a number of cellular processes. To determine whether ATP signaling might be involved in merozoite invasion, we investigated whether known plasmodium invasion proteins contain nucleotide binding motifs. Domain mapping identified a putative nucleotide binding region within all members of the reticulocyte-binding protein homologue (RBL) family analyzed. A representative domain, termed here nucleotide binding domain 94 (NBD94), was expressed and demonstrated to specifically bind to ATP. Nucleotide affinities of NBD94 were determined by fluorescence correlation spectroscopy, where an increase in the binding of ATP is observed compared with ADP analogues. ATP binding was reduced by the known F1F0-ATP synthase inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Fluorescence quenching and circular dichroism spectroscopy of NBD94 after binding of different nucleotides provide evidence for structural changes in this protein. Our data suggest that different structural changes induced by ATP/ADP binding to RBL could play an important role during the invasion process.

Low resolution structure of subunit b (b (22-156)) of Escherichia coli F(1)F(O) ATP synthase in solution and the b-delta assembly.

The first low resolution solution structure of the soluble domain of subunit b (b (22-156)) of the Escherichia coli F(1)F(O) ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 +/- 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit delta, confirming their described neighborhood. Using the recombinant C-terminal domain (delta(91-177)) of subunit delta and the C-terminal peptides of subunit b, b (120-140) and b (140-156), FCS titration experiments were performed to assign the segments involved in delta-b assembly. These data identify the very C-terminal tail b (140-156) to interact with delta(91-177). The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E.

A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.

Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii.

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.

Crystal structure of the NS3 protease-helicase from dengue virus.

Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 A. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain.