Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil.

The Nanoscopium 155 m-long beamline of Synchrotron Soleil is dedicated to scanning hard X-ray nanoprobe techniques. Nanoscopium aims to reach ≤100 nm resolution in the 5-20 keV energy range for routine user experiments. The beamline design tackles the tight stability requirements of such a scanning nanoprobe by creating an overfilled secondary source, implementing all horizontally reflecting main beamline optics, applying high mechanical stability equipment and constructing a dedicated high-stability building envelope. Multi-technique scanning imaging and tomography including X-ray fluorescence spectrometry and spectro-microscopy, absorption, differential phase and dark-field contrasts are implemented at the beamline in order to provide simultaneous information on the elemental distribution, speciation and sample morphology. This paper describes the optical concept and the first measured performance of the Nanoscopium beamline followed by the hierarchical length-scale multi-technique imaging experiments performed with dwell times down to 3 ms per pixel.

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 X-ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging-plate detector and then measured with both the XPAD3S-CdTe and the XPAD3S-Si, a silicon photon-counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit-cell parameter determination and the measurement of high-quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X-ray diffraction opened up by this type of technology are discussed.

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. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.

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. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 A resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 53.78, c = 162.05 A.

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. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production.

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. We propose a model in which the structural constraints of this region control the architecture of the RAR/RXR/TFIIH complex and therefore the efficiency of RARalpha phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex.

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. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.

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 1.75 A resolution of the sensor domain of BlaR1 in complex with a cephalosporin antibiotic. Activation of the signal transducer involves acylation of serine 389 by the beta-lactam antibiotic, a process promoted by the N-carboxylated side chain of Lys392. We present evidence that, on acylation, the lysine side chain experiences a spontaneous decarboxylation that entraps the sensor in its activated state. Kinetic determinations and quantum mechanical/molecular mechanical calculations and the interaction networks in the crystal structure shed light on how this unprecedented process for activation of a receptor may be achieved and provide insights into the mechanistic features that differentiate the signal-transducing receptor from the structurally related class D beta-lactamases, enzymes of antibiotic resistance.

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. Most of these parameters are easy to estimate, and the results of phasing are practically independent of these parameters when they are chosen within reasonable limits. By contrast, the correct choice for such parameters as the expected number of connected regions in the unit cell is sometimes ambiguous. To study these dependencies, numerous tests were performed with simulated data, experimental data and mixed data sets, where several reflections missed in the experiment were completed by computed data. This paper demonstrates that the procedure is able to control this choice automatically and helps in difficult cases to identify the correct number of molecules in the asymmetric unit. In addition, the procedure behaves abnormally if the space group is defined incorrectly and therefore may distinguish between the rotation and screw axes even when high-resolution data are not available.

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. Interestingly, incubation of apoTorA with TorD before cofactor addition led to a significant increase of apoTorA activation, suggesting that TorD acts on apoTorA before cofactor insertion. This result is consistent with the fact that TorD binds to apoTorA and probably modifies its conformation in the absence of cofactor. Therefore, we propose that TorD is involved in the first step of TorA maturation to make it competent to receive the cofactor.

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. The all-helical architecture of the globular domains within the intertwined molecular dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated with the bacterial DMSO/TMAO reductases and also that of proteins of yet unknown functions. The occurrence of multiple oligomeric forms and the chaperone activity of both monomeric and dimeric TorD raise questions about the possible biological role of domain swapping in this protein.

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. These modules have similar overall folds but display remarkable differences in the conformation of the beta4-alpha4 and alpha4 regions. The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region. Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains. The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces. This design is well suited for further association of PhoP dimers to form oligomeric structures. The interprotein interface buries 970 A(2) from solvent and mostly involves interactions between charged residues. As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences. The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.

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 PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoP(R113E), PhoP(R113A), or PhoP(D60K) resulted in a Pho-negative phenotype. In vitro analysis showed that PhoP(R113E) was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoP(R113E) approximately P was deficient in DNA binding, contributing to the PhoP(R113E) in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

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. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed.

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. This motion increases the solvent accessibility of the single cysteine residue in the protein. Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple. Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K(D) values for the catalytic metal ion are coupled events. We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivo through the specific protein-protein interactions between the response regulator and its cognate kinases.

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. The enzyme is as active as the wild-type in turnover of typical beta-lactam antibiotics. Furthermore, many of the parameters for interactions of the inhibitors with the mutant enzyme are largely unaffected. The significant effect of the inhibitor-resistant trait was a relatively modest elevation of the dissociation constant for the formation of the pre-acylation complex. The high-resolution X-ray crystal structure for the M69L mutant variant revealed essentially no alteration of the three-dimensional structure, both for the protein backbone and for the positions of the side chains of the amino acids. It was surmised that the difference in the two enzymes must reside with the dynamic motions of the two proteins. Molecular dynamics simulations of the mutant and wild-type proteins were carried out for 2 ns each. Dynamic cross-correlated maps revealed the collective motions of the two proteins to be very similar, yet the two proteins did not behave identically. Differences in behavior of the two proteins existed in the regions between residues 145-179 and 155-162. Additional calculations revealed that kinetic effects measured experimentally for the dissociation constant for the pre-acylation complex could be mostly attributed to the electrostatic and van der Waals components of the binding free energy. The effects of the mutation on the behavior of the beta-lactamase were subtle, including the differences in the measured dissociation constants that account for the inhibitor-resistant trait. It would appear that nature has selected for incorporation of the most benign alteration in the structure of the wild-type TEM-1 beta-lactamase that is sufficient to give the inhibitor-resistant trait.

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. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.

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 = 37.2, b = 40.5, c = 67.1 A and diffract beyond 1.6 A on a synchrotron beamline.

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. We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative. The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.