Gerhard Grüber

Crystal Structure of the NS3 Protease-Helicase from Dengue Virus

ABSTRACT 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 Å. 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.

A WNK kinase binds and phosphorylates V-ATPase subunit C.

WNK (with no lysine (K)) protein kinases are found in many eukaryotes and share a unique active site. Here, we report that a member of the Arabidopsis WNK family (AtWNK8) interacts with subunit C of the vacuolar H+‐ATPase (V‐ATPase) via a short C‐terminal domain. AtWNK8 is shown to autophosphorylate intermolecularly and to phosphorylate Arabidopsis subunit C (AtVHA‐C) at multiple sites as determined by MALDI‐TOF MS analysis. Furthermore, we show that AtVHA‐C and other V‐ATPase subunits are phosphorylated when V1‐complexes are used as substrates for AtWNK8. Taken together, our results provide evidence that V‐ATPases are potential targets of WNK kinases and their associated signaling pathways.

Eukaryotic V-ATPase: novel structural findings and functional insights.

The eukaryotic V-type adenosine triphosphatase (V-ATPase) is a multi-subunit membrane protein complex that is evolutionarily related to F-type adenosine triphosphate (ATP) synthases and A-ATP synthases. These ATPases/ATP synthases are functionally conserved and operate as rotary proton-pumping nano-motors, invented by Nature billions of years ago. In the first part of this review we will focus on recent structural findings of eukaryotic V-ATPases and discuss the role of different subunits in the function of the V-ATPase holocomplex. Despite structural and functional similarities between rotary ATPases, the eukaryotic V-ATPases are the most complex enzymes that have acquired some unconventional cellular functions during evolution. In particular, the novel roles of V-ATPases in the regulation of cellular receptors and their trafficking via endocytotic and exocytotic pathways were recently uncovered. In the second part of this review we will discuss these unique roles of V-ATPases in modulation of function of cellular receptors, involved in the development and progression of diseases such as cancer and diabetes as well as neurodegenerative and kidney disorders. Moreover, it was recently revealed that the V-ATPase itself functions as an evolutionarily conserved pH sensor and receptor for cytohesin-2/Arf-family GTP-binding proteins. Thus, in the third part of the review we will evaluate the structural basis for and functional insights into this novel concept, followed by the analysis of the potentially essential role of V-ATPase in the regulation of this signaling pathway in health and disease. Finally, future prospects for structural and functional studies of the eukaryotic V-ATPase will be discussed.

Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase.

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).

New insights into structure-function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0).

Adenosine triphosphate, ATP, is the energy currency of living cells. While ATP synthases of archae and ATP synthases of pro‐ and eukaryotic organisms operate as energy producers by synthesizing ATP, the eukaryotic V‐ATPase hydrolyzes ATP and thus functions as energy transducer. These enzymes share features like the hydrophilic catalytic‐ and the membrane‐embedded ion‐translocating sector, allowing them to operate as nano‐motors and to transform the transmembrane electrochemical ion gradient into ATP or vice versa. Since archaea are rooted close to the origin of life, the A‐ATP synthase is probably more similar in its composition and function to the “original” enzyme, invented by Nature billion years ago. On the contrary, the V‐ATPases have acquired specific structural, functional and regulatory features during evolution. This review will summarize the current knowledge on the structure, mechanism and regulation of A‐ATP synthases and V‐ATPases. The importance of V‐ATPase in pathophysiology of diseases will be discussed. BioEssays 30:1096–1109, 2008.

The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Background: NS3-NS5 interaction is important for the dengue virus life cycle. Results: NS3 residue Asn-570 is essential for its interaction with NS5; mutation in an infectious cDNA abolished virus production and reduced positive-strand RNA synthesis. Conclusion: NS3-NS5 interaction may be required for coordinated positive- and negative-strand RNA synthesis. Significance: NS3-NS5 interaction may be a target for rational design of antiviral drugs. Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5′-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566–585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172–618) helicase and covalently linked NS3(172–618)-NS5(320–341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320–341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V‐ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP‐analogues bind more weakly to subunit C than the ADP‐analogues. Site‐directed mutagenesis and N‐terminal sequencing of subunit C from Arabidopsis (VHA‐C) and yeast (Vma5p) have been used to map the C‐terminal region of subunit C as the nucleotide‐binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide‐binding.

Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Vma5p (subunit C) of the yeast V‐ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% α‐helix and 17% β‐sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X‐ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low‐resolution shape of the protein was determined. Vma5p is an elongated boot‐shaped particle consisting of two distinct domains. Co‐reconstitution of Vma5p to V1 without C from Manduca sexta resulted in a V 1–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.

ATP synthases from archaea: the beauty of a molecular motor.

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed.

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli

Hydroperoxides are reactive oxygen species (ROS) that are toxic to all cells and must be converted into the corresponding alcohols to alleviate oxidative stress. In Escherichia coli, the enzyme primarily responsible for this reaction is alkylhydroperoxide reductase (AhpR). Here, the crystal structures of both of the subunits of EcAhpR, EcAhpF (57 kDa) and EcAhpC (21 kDa), have been solved. The EcAhpF structures (2.0 and 2.65 Å resolution) reveal an open and elongated conformation, while that of EcAhpC (3.3 Å resolution) forms a decameric ring. Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. These studies suggest a novel overall mechanistic model of AhpR as a hydroperoxide scavenger, in which the dimeric, extended AhpF prefers complex formation with the AhpC ring to accelerate the catalytic activity and thus to increase the chance of rescuing the cell from ROS.