Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre.

Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Šresolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and ß D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions.

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

Allelic variation of bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels.

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

Metzincin's canonical methionine is responsible for the structural integrity of the zinc-binding site.

The metzincins constitute a subclan of metalloproteases possessing a HEXXHXXGXXH/D zinc-binding consensus sequence where the three histidines are zinc ligands and the glutamic acid is the catalytic base. A completely conserved methionine is located downstream of this motif. Families of the metzincin clan comprise, besides others, astacins, adamalysins proteases, matrix metallo-proteases, and serralysins. The latter are extracellular 50 kDa proteases secreted by Gram-negative bacteria via a type I secretion system. While there is a large body of structural and biochemical information available, the function of the conserved methionine has not been convincingly clarified yet. Here, we present the crystal structures of a number of mutants of the serralysin member protease C with the conserved methionine being replaced by Ile, Ala, and His. Together with our former report on the leucine and cysteine mutants, we demonstrate here that replacement of the methionine side chain results in an increasing distortion of the zinc-binding geometry, especially pronounced in the chi(2) angles of the first and third histidine of the consensus sequence. This is correlated with an increasing loss of proteolytic activity and a sharp increase of flexibility of large segments of the polypeptide chain.

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5.

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.

Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS).

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.

High-resolution structures of Lactobacillus salivarius transketolase in the presence and absence of thiamine pyrophosphate.

Probiotic bacterial strains have been shown to enhance the health of the host through a range of mechanisms including colonization, resistance against pathogens, secretion of antimicrobial compounds and modulation of the activity of the innate immune system. Lactobacillus salivarius UCC118 is a well characterized probiotic strain which survives intestinal transit and has many desirable host-interaction properties. Probiotic bacteria display a wide range of catabolic activities, which determine their competitiveness in vivo. Some lactobacilli are heterofermentative and can metabolize pentoses, using a pathway in which transketolase and transaldolase are key enzymes. L. salivarius UCC118 is capable of pentose utilization because it encodes the key enzymes on a megaplasmid. The crystal structures of the megaplasmid-encoded transketolase with and without the enzyme cofactor thiamine pyrophosphate have been determined. Comparisons with other known transketolase structures reveal a high degree of structural conservation in both the catalytic site and the overall conformation. This work extends structural knowledge of the transketolases to the industrially and commercially important Lactobacillus genus.

The molecular architecture of the metalloprotease FtsH.

The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like sigma(32) and lambda-CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.

Crystal structure of yeast Ypr118w, a methylthioribose-1-phosphate isomerase related to regulatory eIF2B subunits.

Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the alpha-, beta-, and delta-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.