Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins.

BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function in vivo is not yet known. The expression of PEBP and related proteins seems to be correlated with development and cell morphogenesis, however. To obtain new insights into the PEBP family and its potential functions, we initiated a crystallographic study of bovine brain PEPB. RESULTS: The X-ray crystal structure of bovine brain PEBP has been solved using multiple isomorphous replacement methods, and refined to 1.84 A resolution. The structure displays a beta fold and exhibits one nonprolyl cis peptide bond. Analysis of cavities within the structure and sequence alignments were used to identify a putative ligand-binding site. This binding site is defined by residues of the C-terminal helix and the residues His85, Asp69, Gly109 and Tyr119. This site also corresponds to the binding site of phosphorylethanolamine, the polar head group of phosphatidylethanolamine. CONCLUSIONS: This study shows that PEBP is not related to the G-protein family nor to known lipid-binding proteins, and therefore defines a novel structural family of phospholipid-binding proteins. The PEBP structure contains no internal hydrophobic pocket, as described for lipocalins or small phospholipid-transfer proteins. Nevertheless, in PEBP, a small cavity close to the protein surface has a high affinity for anions, such as phosphate and acetate, and also phosphorylethanolamine. We suggest that this cavity corresponds to the binding site of the polar head group of phosphatidylethanolamine.

Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP.

The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms). In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A. The position of the zinc ion has been confirmed and is located near the active site. The tryptic fragment is composed of two globular domains. The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted. The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site. The second domain, up to Pro526, is mainly alpha-helical. The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains. The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located. The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme. The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively. The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose. One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP. The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule. The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features.

The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.

How methionyl-tRNA synthetase creates its amino acid recognition pocket upon L-methionine binding.

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.

Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor protein.

In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.

Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases.

The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.

A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases.

The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.

DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase.

The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.

HU protein of Escherichia coli binds specifically to DNA that contains single-strand breaks or gaps.

In this study, we have identified a protein in Escherichia coli that specifically binds to double-stranded DNA containing a single-stranded gap of one nucleotide. The gap-DNA binding (GDB) protein was purified to apparent homogeneity. The analysis of the amino-terminal sequencing of the GDB protein shows two closely related sequences we identify as the alpha and beta subunits of the HU protein. Furthermore, the GDB protein is not detected in the crude extract of an E. coli double mutant strain hupA hupB that has no functional HU protein. These results led us to identify the GDB protein as the HU protein. HU binds strongly to double-stranded 30-mer oligonucleotides containing a nick or a single-stranded gap of one or two nucleotides. Apparent dissociation constants were measured for these various DNA duplexes using a gel retardation assay. The KD(app) values were 8 nM for the 30-mer duplex that contains a nick and 4 and 2 nM for those that contain a 1-or a 2-nucleotide gap, respectively. The affinity of HU for these ligands is at least 100-fold higher than for the same 30-mer DNA duplex without nick or gap. Other single-stranded breaks or gaps, which are intermediate products in the repair of abasic sites after incision by the Fpg, Nth, or Nfo proteins, are also preferentially bound by the HU protein. Due to specific binding to DNA strand breaks, HU may play a role in replication, recombination, and repair.

AP site structural determinants for Fpg specific recognition.

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.

Crystallization and preliminary X-ray diffraction analysis of the homodimeric form alpha2 of the HU protein from Escherichia coli.

The homodimeric form alpha(2) 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 A, and contain one monomer per asymmetric unit. A full diffraction data set was collected to 2.3 A 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.

Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins.

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.

Structural similarities in glutaminyl- and methionyl-tRNA synthetases suggest a common overall orientation of tRNA binding.

Detailed comparisons between the structures of the tRNA-bound Escherichia coli glutaminyl-tRNA (Gln-tRNA) synthetase [L-glutamine:tRNA(Gln) ligase (AMP-forming), EC 6.1.1.18] and recently refined E. coli methionyl-tRNA (Met-tRNA) synthetase [L-methionine:tRNA(Met) ligase (AMP-forming), EC 6.1.1.10] reveal significant similarities beyond the anticipated correspondence of their respective dinucleotide-fold domains. One similarity comprises a 23-amino acid alpha-helix-turn-beta-strand motif found in each enzyme within a domain that is inserted between the two halves of the dinucleotide binding fold. A second correspondence, which consists of two alpha-helices connected by a large loop and beta-strand, is located in the Gln-tRNA synthetase within a region that binds the inside corner of the "L"-shaped tRNA molecule. This structural motif contains a long alpha-helix, which extends along the entire length of the D and anticodon stems of the complexed tRNA. We suggest that the positioning of this helix relative to the dinucleotide fold plays a critical role in ensuring the proper global orientation of tRNA(Gln) on the surface of the enzyme. The structural correspondences suggest a similar overall orientation of binding of tRNA(Met) and tRNA(Gln) to their respective synthetases.

Crystal structure of a double-stranded DNA containing a cisplatin interstrand cross-link at 1.63 A resolution: hydration at the platinated site.

cis-diamminedichloroplatinum (II) (cisplatin) is a powerful anti-tumor drug whose target is cellular DNA. In the reaction between DNA and cisplatin, covalent intrastrand and interstrand cross-links (ICL) are formed. Two solution structures of the ICL have been published recently. In both models the double-helix is bent and unwound but with significantly different angle values. We solved the crystal structure at 100K of a double-stranded DNA decamer containing a single cisplatin ICL, using the anomalous scattering (MAD) of platinum as a unique source of phase information. We found 47 degrees for double-helix bending and 70 degrees for unwinding in agreement with previous electrophoretic assays. The crystals are stabilized by intermolecular contacts involving two cytosines extruded from the double-helix, one of which makes a triplet with a terminal G.C pair. The platinum coordination is nearly square and the platinum residue is embedded into a cage of nine water molecules linked to the cross-linked guanines, to the two amine groups, and to the phosphodiester backbone through other water molecules. This water molecule organization is discussed in relation with the chemical stability of the ICL.