Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alphabeta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha/beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUα2 and EcHUβ2, and a heterodimer, EcHUαβ. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E. coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N2↔2D), this present thermodynamic study shows that the three E. coli dimers melt according to a two-step mechanism (N2↔I2↔2D). The native dimer, N2, melts partially into a dimeric intermediate, I2, which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUα2 dimer has been solved. Comparative thermodynamic and structural analysis between EcHUα2 and the HU homodimer from Bacillus stearothermophilus suggests that the E. coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I2, corresponds to an HU dimer having partly lost its α-helices. The partially unfolded dimer I2 is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N2↔I2 equilibrium plays a central role in the physiology of E. coli HU. The I2 molecular species seems to be the EcHUβ2 preferential conformation, possibly related to its role in the E. coli cold-shock adaptation. Besides, I2 might be required in E. coli for the HU chain exchange, which allows the heterodimer formation from homodimers.

For protein-DNA complex crystallization, the choice of the DNA fragment is crucial. With the aim of crystallizing the 31 kDa Fpg DNA-repair enzyme bound to DNA, oligonucleotide duplexes varying in length, sequence, end type and nature of the specific DNA target site were used. Crystals of several protein-DNA combinations grew from solutions containing both polyethylene glycol and salt. This systematic crystallization screening followed by optimization of the crystallization conditions by microseeding led to crystals of Fpg bound to a 13 base-pair duplex DNA carrying the 1,3-propanediol abasic site analogue which are suitable for crystallographic analysis. Complete native data sets have been collected to 2.1 A resolution.

Amino acid selection by aminoacyl-tRNA synthetases requires efficient mechanisms to avoid incorrect charging of the cognate tRNAs. A proofreading mechanism prevents Escherichia coli methionyl-tRNA synthetase (EcMet-RS) from activating in vivo L-homocysteine, a natural competitor of L-methionine recognised by the enzyme. The crystal structure of the complex between EcMet-RS and L-methionine solved at 1.8 A resolution exhibits some conspicuous differences with the recently published free enzyme structure. Thus, the methionine delta-sulphur atom replaces a water molecule H-bonded to Leu13N and Tyr260O(eta) in the free enzyme. Rearrangements of aromatic residues enable the protein to form a hydrophobic pocket around the ligand side-chain. The subsequent formation of an extended water molecule network contributes to relative displacements, up to 3 A, of several domains of the protein. The structure of this complex supports a plausible mechanism for the selection of L-methionine versus L-homocysteine and suggests the possibility of information transfer between the different functional domains of the enzyme.

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) catalyses the fourth step of protein synthesis; ribosome recycling.In this step the posttranslocational complex is separated into its components.The crystal structure of Thermotoga maritima ribosome recycling factor (RRF) has been determined to 2.55 Å resolution. 1 RRF overlaps almost perfectly with a tRNA molecule except that the amino acid binding CCA end is missing.The mimicry suggests that RRF binds to the tRNA binding ribosomal A-site and is translocated to the P-site by EF-G.This mechanism is supported by studies of antibiotic action on the RRF reaction.

The homodimeric form α2 of the Escherichia coli DNA-binding protein HU was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belong to space group I222, with unit-cell parameters a = 31.09, b = 55.34, c = 117.63 Å, and contain one monomer per asymmetric unit. A full diffraction data set was collected to 2.3 Å resolution on a conventional X-ray source. The molecular-replacement method, using the HU crystallographic model from Bacillus stearothermophilus as a starting point, gave a reliable solution for the rotation and translation functions.

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.