Modeling of NMR processing, toward efficient unattended processing of NMR experiments.

Many alternative processing techniques have recently been proposed in the literature. Most of these techniques rely on specific acquisition protocols as well as on specific data processing techniques, the need for an efficient versatile and expandable NMR processing tool would be a particularly timely addition to the modern NMR spectroscopy laboratory. The work presented here consists in a modeling of the various possible NMR data processing approaches. This modeling presents a common working frame for most of the modern acquisition/processing protocols. Two different data modeling approaches are presented, strong modeling and weak modeling, depending whether the system under study or the measurement is modeled. The emphasis is placed on the weak modeling approach. This modeling is implemented in a computer program developed in python and called NPK standing (standing for NMR Processing Kernel), organized in four logical layers (i) mathematical kernel; (ii) elementary actions; (iii) processing phases; (iv) processing strategies. This organisation, along with default values for most processing parameters allows the use of the program in an unattended manner, producing close to optimal spectra. Examples are shown for 1D and 2D processing, and liquid and solid NMR spectroscopy. NPK is available from the site: http://abcis.cbs.cnrs.fr/NPK.

kinDOCK: a tool for comparative docking of protein kinase ligands.

KinDOCK is a new web server for the analysis of ATP-binding sites of protein kinases. This characterization is based on the docking of ligands already co-crystallized with other protein kinases. A structural library of protein kinase-ligand complexes has been extracted from the Protein Data Bank (PDB). This library can provide both potential ligands and their putative binding orientation for a given protein kinase. After protein-protein structural superposition, the ligands are transferred from the template complexes to the target protein kinase. The resulting complexes are evaluated using the program SCORE to compute a theoretical affinity. They can be dynamically visualized to allow a rapid mapping of important steric clashes and potential substitutions relevant for specificity and affinity. These characteristics allow a quick characterization of protein kinase active sites including conformation changes potentially required to accommodate particular ligands. Additionally, promising pharmacophores can be identified in the focussed library. These features will help to rationalize or optimize virtual screening (VS) on larger chemical compound libraries. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/kindock/.

The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria.

Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. Here, we demonstrate that mitoxantrone, an anthraquinone derivative used in cancer therapy, is a PknB inhibitor capable of preventing mycobacterial growth. The structure of the complex reveals that mitoxantrone partially occupies the adenine-binding pocket in PknB, providing a framework for the design of compounds with potential therapeutic applications. PknB crystallizes as a 'back-to-back' homodimer identical to those observed in other structures of PknB in complex with ATP analogs. This organization resembles that of the RNA-dependent protein kinase PKR, suggesting a mechanism for kinase activation in mycobacteria.

Internal point mutations of the capsid modify the serotype of Rice yellow mottle virus.

Rice yellow mottle virus is classified in five major serotypes; the molecular diversity of the coat protein (CP) is well established, but the amino acids involved in the recognition by discriminant monoclonal antibodies (MAbs) remain unknown. Reconstruction of a phylogenetic tree and sequence alignment of the CP gene of a sample representative of the continental-large diversity were used to identify 10 serospecific amino acids (i.e., conserved in all isolates belonging to the same serotype and distinct in other serotypes). Positions occupied by serospecific residues were localized on the crystal structure of the CP monomer and on modeled capsomers. Structural, molecular, and serological properties of each serotype were analyzed, and subsequently, hypotheses on the potential role of amino acids in discriminating reactions with antibodies were formulated. The residues 114 and 115 (serospecific of Sr1) and 190 (serospecific of Sr2) were localized on the outer surface of the capsid and might be directly involved in the immunoreactivity with MAb D and MAb A, respectively. In contrast, residues 180 (Sr3) and 178 (Sr5) lay within the inner surface of the capsid. To understand the role of these internal positions in the recognition with the antibodies, two substitutions (T180K and G178D) were introduced in the CP of an infectious clone. These mutations modified the antigenicity with MAb G and MAb E discriminating Sr3 and Sr5, respectively, while the reaction with MAb D remained unaffected. This result suggests an indirect effect of these two internal mutations on local immunostructure while the global structure was maintained.

Structural and functional analysis of the C-terminal STAS (sulfate transporter and anti-sigma antagonist) domain of the Arabidopsis thaliana sulfate transporter SULTR1.2.

The C-terminal region of sulfate transporters from plants and animals belonging to the SLC26 family members shares a weak but significant similarity with the Bacillus sp. anti-anti-sigma protein SpoIIAA, thus defining the STAS domain (sulfate transporter and anti-sigma antagonist). The present study is a structure/function analysis of the STAS domain of SULTR1.2, an Arabidopsis thaliana sulfate transporter. A three-dimensional model of the SULTR1.2 STAS domain was built which indicated that it shares the SpoIIAA folds. Moreover, the phosphorylation site, which is necessary for SpoIIAA activity, is conserved in the SULTR1.2 STAS domain. The model was used to direct mutagenesis studies using a yeast mutant defective for sulfate transport. Truncation of the whole SULTR1.2 STAS domain resulted in the loss of sulfate transport function. Analyses of small deletions and mutations showed that the C-terminal tail of the SULTR1.2 STAS domain and particularly two cysteine residues plays an important role in sulfate transport by SULTR1.2. All the substitutions made at the putative phosphorylation site Thr-587 led to a complete loss of the sulfate transport function of SULTR1.2. The reduction or suppression of sulfate transport of the SULTR1.2 mutants in yeast was not due to an incorrect targeting to the plasma membrane. Both our three-dimensional modeling and mutational analyses strengthen the hypothesis that the SULTR1.2 STAS domain is involved in protein-protein interactions that could control sulfate transport.

ViTO: tool for refinement of protein sequence-structure alignments.

UNLABELLED: ViTO is a graphical application, including an editor, of multiple sequence alignment and a three-dimensional (3D) structure viewer. It is possible to manipulate alignments containing hundreds of sequences and to display a dozen structures. ViTO can handle so-called 'multiparts' alignments to allow the visualization of complex structures (multi-chain proteins and/or small molecules and DNA) and the editing of the corresponding alignment. The 3D viewer and the alignment editor are connected together allowing rapid refinement of sequence-structure alignment by taking advantage of the immediate visualization of resulting insertions/deletions and strict conservations in their structural context. More generally, it allows the mapping of informations about the sequence conservation extracted from the alignment onto the 3D structures in a dynamic way. ViTO is also connected to two comparative modelling programs, SCWRL and MODELLER. These features make ViTO a powerful tool to characterize protein families and to optimize the alignments for comparative modelling. AVAILABILITY: http://bioserv.cbs.cnrs.fr/VITO/DOC/. SUPPLEMENTARY INFORMATION: http://bioserv.cbs.cnrs.fr/VITO/DOC/index.html.