Structural Insights into the Inhibition of Zika Virus NS2B-NS3 Protease by a Small-Molecule Inhibitor

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Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

The Pto gene was derived originally from the wild tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pto and fen. High conservation of genome organization between the two tomato species allowed us to identify the pto and fen alleles from among the cluster of closely related Pto gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Pti1 serine/threonine kinase. However, the pto kinase shows impaired interaction with Pti1 and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto- and Fen-mediated signal transduction pathways.     

Electron Microscopical and Biochemical Characterization of Infectious Pancreatic Necrosis Virus

An electron microscopical and biochemical examination of the properties of infectious pancreatic necrosis virus (IPN) and of its ribonucleic acid (RNA) was made. The buoyant density of IPN in CsCl was found to be 1.33 g/cm³. Electron microscopical examination of the banded virus revealed structures similar in size (74 nm) and shape to reoviruses but lacking a characteristic inner capsid structure. Polyacrylamide gel electrophoretic analysis of IPN-RNA revealed a single non-segmented component of molecular weight 3.2 × 10⁶. Its susceptibility to ribonuclease, base composition, and resistance to thermal denaturation indicated a single-stranded RNA structure. However, its sedimentation behavior (16S) independent of ionic strength in sucrose gradients, partial solubility in 2 m LiCl, and ribonuclease resistance in the presence of Mg²⁺ suggest an unusual secondary structure of unknown nature. The accumulated data indicate that IPN virus does not belong to either the picornavirus or reovirus groups and may represent a new group of viruses.     

Structural Proteins of Reoviruses

Polyacrylamide gel electrophoresis of the solubilized proteins from the three serotypes of reovirus revealed that each contained three major and four minor components. Subviral particles were prepared by brief treatment of complete virions with urea. Electron microscopy, density-gradient centrifugation, and chemical analyses of these particles indicated that their outer capsid structure had been selectively removed. They contained only two proteins, but their ribonucleic acid composition was similar to that of complete virions. The subviral particles were not infectious.     

Structure of the catalytic domain of human polo-like kinase 1

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.     

Backbone dynamics of inactive, active, and effector-bound Cdc42Hs from measurements of (15)N relaxation parameters at multiple field strengths.

Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the beta2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the backbone dynamics of three constructs of [(15)N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using (15)N-(1)H NMR measurements of T(1), T(1)(rho), and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (S(s)(2) and S(f)(2)), local correlation time (tau(e)), and exchange rate (R(ex)) were obtained using the Lipari-Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs-GMPPCP and Cdc42Hs-PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein-protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions.     

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable f ibronectin‐binding, c ollagen‐binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT‐6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain‐of‐function mutants we show that, unlike other GAS pili, the FCT‐6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT‐6 pili.     

Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.