Energy resolution of the CdTe-XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals.

The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL.

The PHD finger of human UHRF1 reveals a new subgroup of unmethylated histone H3 tail readers.

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry.

Expression, purification, crystallization and preliminary crystallographic study of the SRA domain of the human UHRF1 protein.

Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code.

High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively.

Cyclin H binding to the RARalpha activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7.

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARalpha is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of S77 governing the transcriptional activity of RARalpha depends on cyclin H binding at a RARalpha region that encompasses loop 8-9 and the N-terminal tip of helix 9 of the AF-2 domain.

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL.

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity.

X-ray crystal structure of the acylated beta-lactam sensor domain of BlaR1 from Staphylococcus aureus and the mechanism of receptor activation for signal transduction.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are the major cause of infections worldwide. Transcription of the beta-lactamase and PBP2a resistance genes is mediated by two closely related signal-transducing integral membrane proteins, BlaR1 and MecR1, upon binding of the beta-lactam inducer to the sensor domain. Herein we report the crystal structure at 1.

Ab initio phasing based on topological restraints: automated determination of the space group and the number of molecules in the unit cell.

The connectivity-based phasing method has been demonstrated to be capable of finding molecular packing and envelopes even for difficult cases of structure determination, as well as of identifying, in favorable cases, secondary-structure elements of protein molecules in the crystal. This method uses a single set of structure factor magnitudes and general topological features of a crystallographic image of the macromolecule under study. This information is expressed through a number of parameters.

Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA.

As many prokaryotic molybdoenzymes, the trimethylamine oxide reductase (TorA) of Escherichia coli requires the insertion of a bis(molybdopterin guanine dinucleotide)molybdenum cofactor in its catalytic site to be active and translocated to the periplasm. We show in vitro that the purified apo form of TorA was activated weakly when an appropriate bis(molybdopterin guanine dinucleotide)molybdenum source was provided, whereas addition of the TorD chaperone increased apoTorA activation up to 4-fold, allowing maturation of most of the apoprotein. We demonstrate that TorD alone is sufficient for the efficient activation of apoTorA by performing a minimal in vitro assay containing only the components for the cofactor synthesis, apoTorA and TorD.

A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia.

TorD is the cytoplasmic chaperone involved in the maturation of the molybdoenzyme TorA prior to the translocation of the folded protein into the periplasm. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits.

Residue R113 is essential for PhoP dimerization and function: a residue buried in the asymmetric PhoP dimer interface determined in the PhoPN three-dimensional crystal structure.

Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which P(i) is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3' of PhoP-binding consensus repeats in PhoP-repressed promoters.

The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface.

PhoP from Bacillus subtilis belongs to the OmpR subfamily of response regulators. It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency. The receiver domains of two members of this subfamily, PhoB from Escherichia coli and DrrD from Thermotoga maritima, have been structurally characterized.

Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101.

FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations.

Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA.

Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species.

Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus.

DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle. Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded alpha/beta fold of the DivK protein is fully defined only at acidic pH. The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the beta4-alpha4 loop and of the alpha4 helix.

Molecular dynamics at the root of expansion of function in the M69L inhibitor-resistant TEM beta-lactamase from Escherichia coli.

Clavulanate, an inhibitor for beta-lactamases, was the very first inhibitor for an antibiotic resistance enzyme that found clinical utility in 1985. The clinical use of clavulanate and that of sulbactam and tazobactam, which were introduced to the clinic subsequently, has facilitated evolution of a set of beta-lactamases that not only retain their original function as resistance enzymes but also are refractory to inhibition by the inhibitors. This article characterizes the properties of the clinically identified M69L mutant variant of the TEM-1 beta-lactamase from Escherichia coli, an inhibitor-resistant beta-lactamase, and compares it to the wild-type enzyme.

Characterization and crystallization of DivK, an essential response regulator for cell division and differentiation in Caulobacter crescentus.

DivK is an essential response regulator involved in the complex signal transduction network required for cell division and cell differentiation in Caulobacter crescentus. Small-angle X-ray scattering analysis was valuable for obtaining single crystals of the DivK recombinant protein. These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 37.

High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 beta-lactamase.

Beta-lactamases are resistance enzymes for beta-lactam antibiotics. These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. Of the four classes of known beta-lactamases, the enzymes of class D are the least understood.