Advances in computation have been enabling many recent advances in biomolecular applications of NMR. Due to the wide diversity of applications of NMR, the number and variety of software packages for processing and analyzing NMR data is quite large, with labs relying on dozens, if not hundreds of software packages. Discovery, acquisition, installation, and maintenance of all these packages is a burdensome task. Because the majority of software packages originate in academic labs, persistence of the software is compromised when developers graduate, funding ceases, or investigators turn to other projects. To simplify access to and use of biomolecular NMR software, foster persistence, and enhance reproducibility of computational workflows, we have developed NMRbox, a shared resource for NMR software and computation. NMRbox employs virtualization to provide a comprehensive software environment preconfigured with hundreds of software packages, available as a downloadable virtual machine or as a Platform-as-a-Service supported by a dedicated compute cloud. Ongoing development includes a metadata harvester to regularize, annotate, and preserve workflows and facilitate and enhance data depositions to BioMagResBank, and tools for Bayesian inference to enhance the robustness and extensibility of computational analyses. In addition to facilitating use and preservation of the rich and dynamic software environment for biomolecular NMR, NMRbox fosters the development and deployment of a new class of metasoftware packages. NMRbox is freely available to not-for-profit users.

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

The amino-terminal 8-kDa domain of DNA polymerase ␤ functions in binding single-stranded DNA (ssDNA), recognition of a 5-phosphate in gapped DNA structures, and as a 5-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Singlestranded DNA binding was probed by photochemical cross-linking to oligo(dT) 16 . Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5 terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys 35 is involved in 5-phosphate recognition of DNA polymerase ␤. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA.

In addition to large domains, many short motifs mediate functional post-translational modification of proteins as well as protein-protein interactions and protein trafficking functions. We have constructed a motif database comprising 312 unique motifs and a web-based tool for identifying motifs in proteins. Functional motifs predicted by MnM can be ranked by several approaches, and we validated these scores by analyzing thousands of confirmed examples and by confirming prediction of previously unidentified 14-3-3 motifs in EFF-1.

eThe total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. T oll-like receptors (TLRs) represent a diverse family of molecules that play a critical role in activating the innate immune system in response to pathogens (1, 2). Toll-like receptor 2 (TLR2) recognizes diverse molecular structures of microbial cell wall origin, including lipoteichoic acid, lipoproteins, peptidoglycan from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from yeast cell walls. TLR2 is reported to be activated by many other microbial products, including phenol-soluble modulins (3) and Porphyromonas gingivalis lipoprotein (4), lipopolysaccharide (LPS) (5-7), and fimbriae (8-10). However, two recent reports have questioned the extent to which lipoprotein, LPS, or fimbriae mediate TLR2 engagement by P. gingivalis (11,12).We previously reported that the total lipid extract of P. gingivalis promotes activation of mouse dendritic cells and inhibits osteoblast-mediated bone deposition through engagement of TLR2 (13,14). These effects were attributed to the dominant phosphorylated dihydroceramide lipids of P. gingivalis, in particular, phosphoethanolamine dihydroceramides. These studies reported engagement of TLR2 only in vitro in mouse cells. Recent reports have demonstrated TLR2-dependent periodontal bone loss in mice following oral infection with P. gingivalis (15,16). Most recently, cell adhesion mediated through the expression of fimbriae by P. gingivalis has been implicated in promoting of TLR2-dependent oral bone loss (17). In contrast, two recent reports indicated that the capacity of fimbriae to engage TLR2 is dependent on the presence of a contaminating factor that is susceptible to hydrolysis by lipoprotein lipase (11,18).In addition to effects on mouse cells, the phosphorylated dihydroceramide lipids of P. gingivalis have been shown to promote proinflammatory responses in human fibroblasts and to cause disruption of human fibroblast adherence/vitality in culture (19). However, it is not clear whether these effects require engagement of TLR2. Since the total lipid extract of P. gingivalis has been shown to activate TLR2 in mice and in mouse cells, the primary purpose of this investigation was to further identify and characterize the specific lipid classes of P. gingivalis that are responsible for engagement of TLR2 and, specifica...