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1.  The metavinculin tail domain directs constitutive interactions with raver1 and vinculin RNA 
Journal of molecular biology  2012;422(5):10.1016/j.jmb.2012.06.015.
Vinculin is a key regulator of the attachment of the actin cytoskeleton to the cell membrane at cellular adhesion sites that is crucial for processes like cell motility and migration, development, survival, and wound healing. Vinculin loss results in embryonic lethality, cardiovascular diseases, and cancer. Its tail domain, Vt, is crucial for vinculin activation and focal adhesion turnover and binds to the actin cytoskeleton and acidic phospholipids upon which it unfurls. The RNA binding protein raver1 regulates the assembly of focal adhesions transcriptionally by binding to vinculin. The muscle-specific splice form, metavinculin, is characterized by a 68 residue insert in the tail domain (MVt) and correlates with hereditary idiopathic dilated cardiomyopathy. Here we report that metavinculin can bind to raver1 in its inactive state. Our crystal structure explains this permissivity, where an extended coil unique to MVt is unfurled in the MVtΔ954:raver1 complex structure. Our binding assays show that raver1 forms a ternary complex with MVt and vinculin mRNA. These findings suggest that the metavinculin:raver1:RNA complex is constitutively recruited to adhesion complexes.
doi:10.1016/j.jmb.2012.06.015
PMCID: PMC3835166  PMID: 22709580
adherens junction; cardiomyopathy; focal adhesion; RRM domain; RNA binding
2.  Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling 
Journal of Bacteriology  2012;194(18):4837-4846.
The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.
doi:10.1128/JB.00560-12
PMCID: PMC3430337  PMID: 22753070
3.  Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER  
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -­target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
doi:10.1107/S0907444911056058
PMCID: PMC3322596  PMID: 22505257
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
4.  Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in (Hyper)Thermophiles▿† 
Journal of Bacteriology  2011;193(9):2177-2185.
Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent Km values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the Vmax determined at 90°C was 62.9 μmol · min−1 · mg of protein−1. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X4-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.
doi:10.1128/JB.01543-10
PMCID: PMC3133074  PMID: 21378188
5.  Data processing and analysis with the autoPROC toolbox 
Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
doi:10.1107/S0907444911007773
PMCID: PMC3069744  PMID: 21460447
autoPROC; data processing
6.  The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription 
PLoS Pathogens  2010;6(9):e1001038.
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Author Summary
The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
doi:10.1371/journal.ppat.1001038
PMCID: PMC2940758  PMID: 20862324
7.  Crystallization and preliminary X-ray crystallographic studies of human voltage-dependent anion channel isoform I (HVDAC1) 
The human voltage-dependent anion channel was overproduced in bacteria and refolded with the help of detergents. Extensive screening of crystallization conditions resulted in the first crystals to be obtained of this voltage-dependent anion-channel type. The crystals diffracted to a resolution of 3.6 Å.
The major channel by which metabolites can pass through the outer mitochondrial membrane is formed by the voltage-dependent anion-channel (VDAC) family. Functionally, VDAC is involved in the limited exchange of ATP, ADP and small hydrophilic molecules across the outer membrane. Moreover, there is compelling evidence that VDAC isoforms in mammals may act in the cross-talk between mitochondria and the cytoplasm by direct interaction with enzymes involved in energy metabolism and proteins involved in mitochondrial-induced apoptosis. To obtain a high-resolution structure of this channel, human VDAC protein isoform I was overproduced in Escherichia coli. After refolding and testing the correct fold using circular dichroism, a subsequent broad-range screening in different detergents resulted in a variety of crystals which diffracted to 3.5 Å resolution. The crystal lattice belongs to the trigonal space group P321, with unit-cell parameters a = 78.9, c = 165.7 Å and one monomer in the asymmetric unit.
doi:10.1107/S174430910801676X
PMCID: PMC2443964  PMID: 18607100
voltage-dependent anion channel; porins
8.  The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes 
PLoS Pathogens  2009;5(5):e1000428.
Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
Author Summary
The genome of the SARS coronavirus codes for 16 non-structural proteins that are involved in replicating this huge RNA (approximately 29 kilobases). The roles of many of these in replication (and/or transcription) are unknown. We attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by X-ray crystallography. Non-structural protein 3 contains at least seven different functional modules within its 1922-amino-acid polypeptide chain. One of these is the so-called SARS-unique domain, a stretch of about 338 residues that is completely absent from any other coronavirus. It may thus be responsible for the extraordinarily high pathogenicity of the SARS coronavirus, compared to other viruses of this family. We describe here the three-dimensional structure of the SARS-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. Furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called G-quadruplexes). SUD may be involved in binding to viral or host-cell RNA bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell.
doi:10.1371/journal.ppat.1000428
PMCID: PMC2674928  PMID: 19436709
9.  Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor 
PLoS Biology  2008;6(9):e227.
The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 Å crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.
Author Summary
Unlike other classes of receptors, nuclear receptors can bind directly to DNA and act as transcription factors, playing key roles in embryonic development and cellular metabolism. Most nuclear receptors are activated by signal-triggering molecules (ligands) and can regulate their activity by recruiting coactivator proteins. However, the ligands are unknown for a subset of “orphan” nuclear receptors, including the chicken ovalbumin promoter-transcription factors (COUP-TFI and II, and EAR2). COUP-TFs are the most conserved nuclear receptors, with roles in angiogenesis, neuronal development, organogenesis, and metabolic homeostasis. Here we demonstrate that COUP-TFII is a ligand-regulated nuclear receptor that can be activated by unphysiological micromolar concentrations of retinoic acids. We determined the structure of the ligand-free ligand-binding domain of the human COUP-TFII, revealing the autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. These results suggest a mechanism where ligands activate COUP-TFII by releasing the receptor from the autorepressed conformation. The identification of COUP-TFII as a low-affinity retinoic acid receptor suggests ways of searching for the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
Structural and functional studies reveal that the orphan nuclear receptor COUP-TFII is a low-affinity receptor for retinoic acids. paving the way to finding the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
doi:10.1371/journal.pbio.0060227
PMCID: PMC2535662  PMID: 18798693
11.  THE CRYSTAL STRUCTURE OF IRON-FREE HUMAN SERUM TRANSFERRIN PROVIDES INSIGHT INTO INTER-LOBE COMMUNICATION AND RECEPTOR BINDING* 
The Journal of biological chemistry  2006;281(34):24934-24944.
Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependant process. The binding and release of iron results in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF) which was independently determined by two methods: (1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7 Å resolution using a MAD phasing strategy, by substituting the nine methionines in hTF with selenomethionine and (2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human TF and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4° and 49.5° rotations are required to open the N- and C-lobe, respectively, (compared to closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.
doi:10.1074/jbc.M604592200
PMCID: PMC1895924  PMID: 16793765
12.  Structural Dynamics of α-Actinin-Vinculin Interactions†  
Molecular and Cellular Biology  2005;25(14):6112-6122.
α-Actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. α-Actinin interacts with vinculin through the binding of an α-helix (αVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:αVBS structure suggests that αVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. αVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, αVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of αVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and α-actinin binding in focal adhesions versus adherens junctions, respectively.
doi:10.1128/MCB.25.14.6112-6122.2005
PMCID: PMC1168820  PMID: 15988023

Results 1-12 (12)