Bacterial structural genomics initiative: overview of methods and technologies applied to the process of structure determination.

The focus over the last several years on increasing the number of three-dimensional structures of macromolecules by implementation of high throughput methodology has led to the establishment of dedicated structural genomics programs around the world. These worldwide efforts have in turn led to development of novel, parallelized approaches to cloning, expression, purification, and crystallization of proteins. This chapter describes in some detail the approaches and protocols that have been implemented in the Bacterial Structural Genomics Initiative.

Molecular dynamics-solvated interaction energy studies of protein-protein interactions: the MP1-p14 scaffolding complex.

Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energy functions and two different single-structure representations of the complex. Refined binding affinity predictions for select hot-spot mutations are carried out by applying first-principle methods such as the molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) to the molecular dynamics (MD) trajectories for mutated and wild-type complexes.

Structural snapshots of Escherichia coli histidinol phosphate phosphatase along the reaction pathway.

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution.

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD).

The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains.

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases.

Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris.

XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gelfiltration chromatography analyses have suggested that it forms a multimer.

Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris.

The last ORF of an xps gene cluster, designated xpsD, is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues.