Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.     

Crystal structure of a conserved hypothetical protein from Escherichia coli

The crystal structure of a conserved hypothetical protein from Escherichia coli has been determined using X-ray crystallography. The protein belongs to the Cluster of Orthologous Group COG1553 (National Center for Biotechnology Information database, NLM, NIH), for which there was no structural information available until now. Structural homology search with DALI algorism indicated that this protein has a new fold with no obvious similarity to those of other proteins with known three-dimensional structures. The protein quaternary structure consists of a dimer of trimers, which makes a characteristic cylinder shape. There is a large closed cavity with approximate dimensions of 16 Å × 16 Å × 20 Å in the center of the hexameric structure. Six putative active sites are positioned along the equatorial surface of the hexamer. There are several highly conserved residues including two possible functional cysteines in the putative active site. The possible molecular function of the protein is discussed.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl-tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl-tRNA hydrolase could be solved at 1.2 A resolution. It indicates a single alpha/beta globular domain built around a twisted mixed beta-sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica. This similarity allowed the characterization by site-directed mutagenesis of several residues of the active site of peptidyl-tRNA hydrolase. These residues, strictly conserved among the known peptidyl-tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C-end of a neighbouring peptidyl-tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl-tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl-tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.     

Crystal structure of himalayan mistletoe ribosome-inactivating protein reveals the presence of a natural inhibitor and a new functionally active sugar-binding site

Ribosome-inactivating proteins (RIPs) are toxins involved in plant defense. How the plant prevents autotoxicity is not yet fully understood. The present study is the first structural evidence of a naturally inhibited form of RIP from a plant. Himalayan mistletoe RIP (HmRIP) was purified from Viscum album leaves and crystallized with lactose. The structure was determined by the molecular replacement method and refined at 2.8-A resolution. The crystal structure revealed the presence of high quality non-protein electron density at the active site, into which a pteridine derivative (2-amino 4-isopropyl 6-carboxyl pteridine) was modeled. The carboxyl group of the ligand binds strongly with the key active site residue Arg(162), nullifies the positive charge required for catalysis, and thereby acts as a natural inhibitor. Lectin subunits of RIPs have two active sugar-binding sites present in 1alpha- and 2gamma-subdomains. A third functionally active site has been identified in the 1beta-subdomain of HmRIP. The 1beta-site is active despite the absence of conserved polar sugar-binding residues. Loss of these residues is compensated by the following: (i) the presence of an extended site where the penultimate sugar also interacts with the protein; (ii) the interactions of galactose with the protein main chain carbonyl and amide nitrogen atoms; (iii) the presence of a well defined pocket encircled by four walls; and (iv) a favorable stacking of the galactose ring with Tyr(66) besides the conserved Phe(75). The mode of sugar binding is also distinct at the 1alpha and 2gamma sugar-binding sites.     

Crystal structure of human copper homeostasis protein CutC reveals a potential copper-binding site

Copper is an essential trace element to life and particularly plays a pivotal role in the physiology of aerobic organisms. The Cut protein family is associated with copper homeostasis and involved in uptake, storage, delivery, and efflux of copper. CutC is a member of the Cut family and is suggested to be involved in efflux trafficking of cuprous ion. We report here the biochemical and structural characterization of human CutC (hCutC). hCutC can bind Cu(I) with a stoichiometry of 1:1 and an apparent dissociation constant of 15.5 ± 2.8 μM. hCutC assumes a typical TIM-barrel fold and forms a tetramer in both crystal structure and solution which is different from the dimeric architecture of the bacterial CutC. Structure analysis and sequence comparison of CutC proteins from different species reveal two strictly conserved Cys residues on the inner surface of the C-terminal end of the TIM-barrel. Mutations of the two Cys residues can significantly impair the binding ability of hCutC with Cu(I). Our results suggest that hCutC functions as an enzyme with Cu(I) as a cofactor rather than a copper transporter and the potential Cu(I)-binding site consists of the two Cys residues and other conserved residues in the vicinity.     

The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites

The mRNA of Escherichia coli yedU gene is induced 31-fold upon heat shock. The 31-kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 A resolution. YedU monomer has an alpha/beta/alpha sandwich domain and a small alpha/beta domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate-binding site responsible for the chaperone activity. The structure also reveals a potential protease-like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2-His-1-carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.     

Crystal structure of the zinc-binding transport protein ZnuA from Escherichia coli reveals an unexpected variation in metal coordination

Bacterial ATP-binding cassette transport systems for high-affinity uptake of zinc and manganese use a cluster 9 solute-binding protein. Structures of four cluster 9 transport proteins have been determined previously. However, the structural determinants for discrimination between zinc and manganese remain under discussion. To further investigate the variability of metal binding sites in bacterial transporters, we have determined the structure of the zinc-bound transport protein ZnuA from Escherichia coli to 1.75 A resolution. The overall structure of ZnuA is similar to other solute-binding transporters. A scaffolding alpha-helix forms the backbone for two structurally related globular domains. The metal-binding site is located at the domain interface. The bound zinc ion is coordinated by three histidine residues (His78, His161 and His225) and one glutamate residue (Glu77). The functional role of Glu77 for metal binding is unexpected, because this residue is not conserved in previously determined structures of zinc and manganese-specific transport proteins. The observed metal coordination by four protein residues differs significantly from the zinc-binding site in the ZnuA transporter from Synechocystis 6803, which binds zinc via three histidine residues. In addition, the E. coli ZnuA structure reveals the presence of a disulfide bond in the C-terminal globular domain that is not present in previously determined cluster 9 transport protein structures.     

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.     

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.4 Angstroms resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383-397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel beta-sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site.     

Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft

Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking. To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations. Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms. The location of this carboxyl-terminal segment appears to interfere with the binding of double-stranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins.     

Crystal structure of cGMP-dependent protein kinase reveals novel site of interchain communication

The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory, and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here, we report the 2.5?? crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full-length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.     

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.     

Crystal structure of PriB, a primosomal DNA replication protein of Escherichia coli

PriB is one of the Escherichia coli varphiX-type primosome proteins that are required for assembly of the primosome, a mobile multi-enzyme complex responsible for the initiation of DNA replication. Here we report the crystal structure of the E. coli PriB at 2.1 A resolution by multi-wavelength anomalous diffraction using a mercury derivative. The polypeptide chain of PriB is structurally similar to that of single-stranded DNA-binding protein (SSB). However, the biological unit of PriB is a dimer, not a homotetramer like SSB. Electrophoretic mobility shift assays demonstrated that PriB binds single-stranded DNA and single-stranded RNA with comparable affinity. We also show that PriB binds single-stranded DNA with certain base preferences. Based on the PriB structural information and biochemical studies, we propose that the potential tetramer formation surface and several other regions of PriB may participate in protein-protein interaction during DNA replication. These findings may illuminate the role of PriB in varphiX-type primosome assembly.