Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site.

Highlights d Cycloheximide (CHX) prevents mitotic entry right up until the G2/M transition d Inhibition of Wee1 or Wee1/Myt1 can overcome a cycloheximide-induced G2 arrest d Activation of p38 MAPK, not lack of protein synthesis, accounts for the CHX arrest d Mitotic entry in the absence of protein synthesis causes delays in mitotic progression

1 Protein synthesis inhibitors (e.g. cycloheximide) prevent cells from entering mitosis, 2 suggesting that cell cycle progression requires protein synthesis until right before mitotic 3 entry. However, cycloheximide is also known to activate p38 MAPK, which can delay 4 mitotic entry through a G2/M checkpoint. Here we asked whether checkpoint activation 5 or a requirement for protein synthesis is responsible for the cycloheximide effect. We 6 found that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 phase, 7and that G2 duration is normal in about half of these cells. The Wee1/Myt1 inhibitor 8 PD0166285 also prevents cycloheximide from blocking mitotic entry, raising the 9 possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, 10 the ultimate trigger for mitotic entry appears not to be the continued synthesis of mitotic 11 cyclins or other proteins. However, M-phase progression was delayed in cycloheximide-12 plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein 13 synthesis for timely entry and completion of mitosis. 14 15 Impact statement (30 words): 16 Cycloheximide arrests cells in G2 phase due to activation of p38 MAPK, not inhibition of 17 protein synthesis, arguing that protein synthesis in G2 phase is not required for mitotic 18 entry. 19 4 end of G2 phase (12, 22). Cdk1-cyclin B1 then translocates from the cytoplasm to the 42 nucleus just prior to nuclear envelope breakdown (16,(23)(24)(25)(26). 43The final increase in cyclin B1-Cdk1 activity, and decrease in PP2A-B55 activity, is 44 thought to be due to the flipping of two bistable switches. Two feedback loops, a double-45 negative feedback loop involving the Cdk1-inhibitory kinases Wee1/Myt1 and a positive 46 feedback loop involving the Cdk1-activating phosphatase Cdc25, keep Cdk1 activity low 47