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RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination. [ABSTRACT FROM AUTHOR]

Klebsiella oxytoca is a pathogen that causes serious infections in hospital patients. It shows resistance to many clinically used β-lactam antibiotics by producing chromosomally encoded OXY-family β-lactamases. Here, the crystal structure of an OXY-family β-lactamase, OXY-1-1, determined at 1.93 Å resolution is reported. The structure shows that the OXY-1-1 β-lactamase has a typical class A β-lactamase fold and exhibits greater similarity to CTX-M-type β-lactamases than to TEM-family or SHV-family β-lactamases. It is also shown that the enzyme provides more space around the active cavity for the R1 and R2 substituents of β-lactam antibiotics. The half-positive/half-negative distribution of surface electrostatic potential in the substrate-binding pocket indicates the preferred properties of substrates or inhibitors of the enzyme. The results reported here provide a structural basis for the broadened substrate profile of the OXY-family β-lactamases. [ABSTRACT FROM AUTHOR]

Abstract: Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. Structured summary: MINT:6700314: GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114) [Copyright &y& Elsevier]

Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2′-5′ RNA-ligase family. It contains 183 residues and two copies of the H XT X sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2′-5′ RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified to homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 Å and belonged to space group P212121, with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 Å. [ABSTRACT FROM AUTHOR]

The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni2+-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P21, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°. [ABSTRACT FROM AUTHOR]

Bar-headed goose fluoromethaemoglobin (fluoromet-Hb) complexed with inositol hexaphosphate (IHP) has been crystallized using PEG 6000 as precipitant. The crystal belongs to space group P2!1@, with unit-cell parameters a = 59.8, b = 72.0, c = 79.8 Å, β = 102.1°, and diffracts to 2.5 Å resolution. To prove the presence of IHP, the structure was determined by the molecular-replacement method. IHP was observed at the entrance to the central cavity between the N and C termini of two β subunits. [ABSTRACT FROM AUTHOR]

Glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (FTHF) to glycinamide ribonucleotide (GAR), which is an essential step in the de novo synthesis pathway of purines. In Bacillus subtilis, GART is encoded by the gene purN. In order to study the structure and function of B. subtilis GART, the purN gene was amplified, cloned into an expression vector and expressed in soluble form in Escherichia coli. The protein was purified to homogeneity and crystals suitable for X-ray data collection were obtained. These crystals diffracted to 2.5 A resolution and belonged to space group P3(1)21, with unit-cell parameters a = b = 95.5, c = 64.0 A.

SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni2+-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 A resolution diffraction data set was collected using an in-house chromium radiation source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 A, alpha = beta = gamma = 90 degrees.

The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A, beta = 98.82 degrees.

The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 A.

D-Alanine-D-alanine ligase is encoded by the gene ddl (SMU_599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 A. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 79.50, c = 108.97 A. There is one molecule per asymmetric unit.

Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of D-glucosamine 6-phosphate to form N-acetyl-D-glucosamine 6-phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni(2+)-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 A resolution. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 50.08, c = 142.88 A.

Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2'-5' RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 A and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 A, beta = 113.56 degrees.

OXY-1a is an extended-spectrum beta-lactamase from the conditional pathogenic bacterium Klebsiella oxytoca. OXY-1a is responsible for the antibiotic resistance of this pathogen. A soluble form of OXY-1a with a His tag at its C-terminus was overexpressed in Escherichia coli. The recombinant protein was purified and crystallized at room temperature using PEG 4000 as the main precipitant. Two crystal forms were obtained from the same growth conditions. One was orthorhombic, with crystals that diffracted to better than 1.9 A, while the other was tetragonal, with crystals that only diffracted to about 3.0 A. Complete data sets were collected from both crystal forms. The orthorhombic crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.54, b = 73.43, c = 84.56 A, while the tetragonal crystal has unit-cell parameters a = b = 73.72, c = 96.81 A. The asymmetric unit of the orthorhombic crystal is estimated to contain one OXY-1a molecule, giving a crystal volume per protein weight (V(M)) of 2.25 A(3) Da(-1) and a solvent content of 45%.

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 A data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.