The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation

Protein synthesis inhibitors have long been known to prevent G2 phase cells from entering mitosis. Lockhead et al. demonstrate that this G2 arrest is due to the activation of p38 MAPK, not insufficient protein synthesis, arguing that protein synthesis in G2 phase is not absolutely required for mitotic entry

Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization

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

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

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

Data collection and refinement statistics for methylglyoxal reductase (YghZ).

<p>*Values in parentheses are for highest-resolution shell.</p

Proteome fractionation and purification flow chart.

<p>Approximately 500 g of <i>E. coli</i> cells were lysed at pH 7 using a microfluidizer and the cell debris pelleted. The supernatant was applied to a tangential flow column with a nominal molecular weight cut off of 500 kDa, generating 2 fractions (retentate and flow through). The fraction above 500 kDa (retentate) was further purified via sucrose gradients, size exclusion, and ion exchange chromatography prior to crystallization trials. The fraction less than 500 kDa was applied to multiple affinity and ion exchange columns followed by phenyl sepharose, ion exchange, and size exclusion prior to crystallization trials in microfluidic chips.</p

Data collection and refinement statistics for Glucose-6-phosphate isomerase (pGI).

<p>*Values in parentheses are for highest-resolution shell.</p

Crystallization of native source <i>E. coli</i> proteins.

<p>(A) Capillary electrophoresis of purified protein fractions. White stars indicate samples successfully crystallized and black stars represent solved structures. (B) Crystals of 5-keto-4-deoxyuronate isomerase crystallized from fractions of varying purity. Crystal quality was not always correlated with sample purity. (C) Resolution of the data collected versus percent purity of the starting sample based on quantification of protein concentrations by capillary gel electrophoresis with the Caliper system. Sample purity did not correlate with higher resolution data.</p

Data collection and refinement statistics for glutamate dehydrogenase (GDH).

<p>Data collection and refinement statistics for glutamate dehydrogenase (GDH).</p

BglA dimer and putative active site.

<p>Left, BglA dimer with the putative active site outlined in a gray box. Right, close up of the active site with glucose-6-phosphate modeled based of the position of the sulfate ion from crystallization. Active site residues are depicted as ball-and-stick. Putative hydrogen bonds to the substrate are drawn as dashed lines.</p