The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule-or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates ␤-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of ␤-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-␤3-tubulin S172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to ␤-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis. INTRODUCTIONIn eukaryotic cycling cells, interphase microtubules form a dynamic network that is essential for cell polarity and intracellular traffic. When cells enter into mitosis, the interphase microtubule network rearranges into a mitotic spindle that is responsible for faithful chromosome segregation between daughter cells. Microtubule dynamics and turnover increase strikingly as cells progress from interphase to mitosis, with a microtubule half-life of 5-10 min in interphase and of 60 -90 s in mitosis (Wittmann et al., 2001).Microtubule dynamics in cells rely in part on the intrinsic properties of the microtubule building block, the ␣-␤-tubulin dimer, and its ability to bind and hydrolyze a GTP nucleotide (Mitchison and Kirschner, 1984). The tubulin dimer is subject to special posttranslational modifications such as glutamylation, tyrosination, and acetylation (MacRae, 1997;Westermann and Weber, 2003). There is little evidence for a role of these modifications in the regulation of microtubule dynamics. Tubulin can also be phosphorylated by several kinases (Westermann and Weber, 2003). However, tubulin phosphorylation has not been connected with the cell-cycle-dependent regulation of microtubule dynamics.Microtubule dynamics are also regulated by a number of microtubule effectors, including microtubule-associated proteins (MAPs), molecular motors such as kinesins, the Raslike GTPase Ran-GTP, microtubule plus end-directed proteins and tubulin-binding proteins (Andersen, 1999(Andersen, , 2000Carazo-Salas et al., 2001;Cassimeris, 2002;Heald and Nogales, 2002;Kinoshita et al., 2002;Galjart and Perez, 2003). These microtubule-or tubulin-associated proteins are themselves under the control of a...

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

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The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with ∼12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases.

The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-L-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle.

The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1 00 -phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182-355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2 1 , with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 Å , = 91.4, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 Å .