Structures of the substrate-binding protein YfeA in apo and zinc-reconstituted holo forms.

In the structural biology of bacterial substrate-binding proteins (SBPs), a growing number of comparisons between substrate-bound and substrate-free forms of metal atom-binding (cluster A-I) SBPs have revealed minimal structural differences between forms. These observations contrast with SBPs that bind substrates such as amino acids or nucleic acids and may undergo >60° rigid-body rotations. Substrate transfer in these SBPs is described by a Venus flytrap model, although this model may not apply to all SBPs. In this report, structures are presented of substrate-free (apo) and reconstituted substrate-bound (holo) YfeA, a polyspecific cluster A-I SBP from Yersinia pestis. It is demonstrated that an apo cluster A-I SBP can be purified by fractionation when co-expressed with its cognate transporter, adding an alternative strategy to the mutagenesis or biochemical treatment used to generate other apo cluster A-I SBPs. The apo YfeA structure contains 111 disordered protein atoms in a mobile helix located in the flexible carboxy-terminal lobe. Metal binding triggers a 15-fold reduction in the solvent-accessible surface area of the metal-binding site and reordering of the 111 protein atoms in the mobile helix. The flexible lobe undergoes a 13.6° rigid-body rotation that is driven by a spring-hammer metal-binding mechanism. This asymmetric rigid-body rotation may be unique to metal atom-binding SBPs (i.e. clusters A-I, A-II and D-IV).

Structures of soluble rabbit neprilysin complexed with phosphoramidon or thiorphan.

Neutral endopeptidase (neprilysin; NEP) is a proteinase that cleaves a wide variety of peptides and has been implicated in Alzheimer's disease, cardiovascular conditions, arthritis and other inflammatory diseases. The structure of the soluble extracellular domain (residues 55-750) of rabbit neprilysin was solved both in its native form at 2.1 Šresolution, and bound to the inhibitors phosphoramidon and thiorphan at 2.8 and 3.0 Šresolution, respectively. Consistent with the extracellular domain of human neprilysin, the structure reveals a large central cavity which contains the active site and the location for inhibitor binding.

Bovine herpesvirus-1 VP8 interacts with DNA damage binding protein-1 (DDB1) and is monoubiquitinated during infection.

VP8 is the most abundant tegument protein of bovine herpesvirus-1 (BHV-1). In the present study DNA damage binding protein 1 (DDB1) was identified as interacting partner of VP8. MALDI-TOF mass spectroscopy analysis of proteins co-immunoprecipitated with VP8 identified DDB1 as a protein interacting with VP8. The interaction between VP8 and DDB1 was confirmed based on co-immunoprecipitation and co-localization in both VP8-transfected and BHV-1 infected cells. DDB1 was distributed both in the nucleus and the cytoplasm with some nuclear speckles prior to BHV-1 infection, became perinuclear by 4h and was predominantly nuclear at 5h post infection, where it co-localized with VP8. In contrast, in cells infected with a U(L)47 deletion mutant DDB1 remained cytoplasmic throughout the course of infection. This suggests that VP8 mediates nuclear re-localization of DDB1. Finally, VP8 was shown to be monoubiquitinated both in VP8-transfected and BHV-1-infected cells. These data suggest that BHV-1 VP8 interacts with DDB1-CUL4 E3 ubiquitin ligase, which correlates to monoubiquitination of this viral protein.

Beamline 08ID-1, the prime beamline of the Canadian Macromolecular Crystallography Facility.

Beamline 08ID-1 is the prime macromolecular crystallography beamline at the Canadian Light Source. Based on a small-gap in-vacuum undulator, it is designed for challenging projects like small crystals and crystals with large cell dimensions. Beamline 08ID-1, together with a second bending-magnet beamline, constitute the Canadian Macromolecular Crystallography Facility (CMCF). This paper presents an overall description of the 08ID-1 beamline, including its specifications, beamline software and recent scientific highlights. The end-station of the beamline is equipped with a CCD X-ray detector, on-axis crystal visualization system, a single-axis goniometer and a sample automounter allowing remote access to the beamline. The general user program is guaranteed up to 55% of the useful beam time and is run under a peer-review proposal system. The CMCF staff provide `Mail-in' crystallography service to the users with the highest-scored proposals.

Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22.

The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases.

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG-VP8 was expressed alone or co-expressed with US3-haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

Cobalt(II), nickel(II) and zinc(II) do not bind to intra-helical N(7) guanine positions in the B-form crystal structure of d(GGCGCC).

Three novel X-ray crystal structures for the DNA hexamer d(GGCGCC) in the B-form complexed to divalent cobalt, nickel and zinc ions have been determined to a resolution of 2.9-3.0 A. The structures were isomorphous and had five DNA strands and five metal cations per asymmetric unit. In all three cases, divalent metal cations were coordinated only to the terminal guanine residue at the N(7) position, with no metal ions binding to non-terminal guanine positions. Water molecules bound to the metal cations interacted with neighboring guanine residues 3' to the ones to which the cations were coordinated, affecting the propeller twist. Even though DNA occupied only about 35% of the unit cell volume, it is interesting that the few interactions involving the metal cations were sufficient to stabilize the crystal lattice. As well as lending support to the proposal that these metals do not coordinate to B-DNA in a stable manner, the results presented here also extend the crystallographic evidence for this phenomenon to the GGC and CGC sequences for all three metal cations.

Jel44 monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and the complex of Jel44 Fab fragment with HPr: preparation, crystallization and preliminary crystallographic analysis.

Jel44 is a mouse monoclonal antibody specific for the histidine-containing phosphocarrier protein (HPr), a component of a sugar-transport system in Escherichia coli. Because Jel44 binding to HPr is dependent upon ionic strength and the enthalpic and entropic contributions do not vary over the temperature range 277-310 K, the complex is of great interest. A single crystal of the Jel44 Fab fragment was obtained and diffracted X-rays to a maximum resolution of 4.6 A on an in-house X-ray source. The crystal belongs to space group P2(1), with unit-cell parameters a = 68.6, b = 67.7, c = 105.5 A, beta = 96 degrees. Although crystals of the complex of Jel44 Fab fragment with HPr could not be fully characterized owing to suspected crystal twinning, it was encouraging that they diffracted X-rays to 2.5 A on an in-house X-ray source. It is thus foreseen that improvement of crystal quality will allow the complete solution of this novel structure.