Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus.

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.

The origin of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity.

Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases.

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.

Structure of Xanthomonas axonopodis pv. citri YaeQ reveals a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif.

The YaeQ family of proteins are found in many Gram-negative and a few Gram-positive bacteria. We have determined the first structure of a member of the YaeQ family by X-ray crystallography. Comparisons with other structures indicate that YaeQ represents a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif found in type II endonucleases and enzymes involved in DNA replication, repair, and recombination. We show that catalytically important residues in the PD-(D/E)XK nuclease superfamily are spatially conserved in YaeQ and other highly conserved YaeQ residues may be poised to interact with nucleic acid structures.

Expression, crystallization and preliminary crystallographic analysis of SufE (XAC2355) from Xanthomonas axonopodis pv. citri.

Xanthomonas axonopodis pv. citri (Xac) SufE (XAC2355) is a member of a family of bacterial proteins that are conserved in several pathogens and phytopathogens. The Escherichia coli suf operon is involved in iron-sulfur cluster biosynthesis under iron-limitation and stress conditions. It has recently been demonstrated that SufE and SufS form a novel two-component cysteine desulfarase in which SufS catalyses the conversion of L-cysteine to L-alanine, forming a protein-bound persulfide intermediate. The S atom is then transferred to SufE, from which it is subsequently transferred to target molecules or reduced to sulfide in solution. Here, the cloning, expression, crystallization and phase determination of Xac SufE crystals are described. Recombinant SufE was crystallized in space group P2(1)2(1)2(1) and diffracted to 1.9 A resolution at a synchrotron source. The unit-cell parameters are a = 45.837, b = 58.507, c = 98.951 A, alpha = beta = gamma = 90 degrees. The calculated Matthews coefficient indicated the presence of two molecules in the asymmetric unit. Phasing was performed by molecular-replacement using E. coli SufE as a model (PDB code 1mzg) and an interpretable map was obtained.

Expression, purification, crystallization and preliminary X-ray analysis of YaeQ (XAC2396) from Xanthomonas axonopodis pv. citri.

Xanthomonas axonopodis pv. citri YaeQ (XAC2396) is a member of a family of bacterial proteins conserved in several Gram-negative pathogens. Here, the cloning, expression, purification and crystallization of the 182-residue (20.6 kDa) YaeQ protein are described. Recombinant YaeQ containing selenomethionine was crystallized in space group P2(1) and crystals diffracted to 1.9 A resolution at a synchrotron source. The unit-cell parameters are a = 39.75, b = 91.88, c = 48.03 A, beta = 108.37 degrees. The calculated Matthews coefficient suggests the presence of two YaeQ molecules in the asymmetric unit. Initial experimental phases were calculated by the multiple-wavelength anomalous dispersion technique and an interpretable electron-density map was obtained.

Crystal structure of a trimeric form of dephosphocoenzyme A kinase from Escherichia coli.

Coenzyme A (CoA) is an essential cofactor used in a wide variety of biochemical pathways. The final step in the biosynthesis of CoA is catalyzed by dephosphocoenzyme A kinase (DPCK, E.C. 2.7.1.24). Here we report the crystal structure of DPCK from Escherichia coli at 1.8 A resolution. This enzyme forms a tightly packed trimer in its crystal state, in contrast to its observed monomeric structure in solution and to the monomeric, homologous DPCK structure from Haemophilus influenzae. We have confirmed the existence of the trimeric form of the enzyme in solution using gel filtration chromatography measurements. Dephospho-CoA kinase is structurally similar to many nucleoside kinases and other P-loop-containing nucleotide triphosphate hydrolases, despite having negligible sequence similarity to these enzymes. Each monomer consists of five parallel beta-strands flanked by alpha-helices, with an ATP-binding site formed by a P-loop motif. Orthologs of the E. coli DPCK sequence exist in a wide range of organisms, including humans. Multiple alignment of orthologous DPCK sequences reveals a set of highly conserved residues in the vicinity of the nucleotide/CoA binding site.

Substrate-Assisted Catalysis in Sialic Acid Aldolase.

Sialic acid aldolase catalyses the reversible aldol condensation of pyruvate and N-acetylmannosamine with an apparent lack of stereospecificity. Consistent with this, modeling of Schiff base and enamine intermediates in the active site of this enzyme yields two conformations, corresponding to si- and re-face attack in the aldol condensation reaction. The acceptor-aldehyde group is found on different sides of the enamine in the two conformations, but with the remainder of the substrate having very similar geometries in the protein. No histidine residue previously speculated to function as a general base in the mechanism is found near the enzyme active site. In the absence of functionally active groups in the active site, the carboxylate of the substrate is proposed to function as the general acid/base. Molecular orbital calculations indicate that the barrier to aldol cleavage via this mechanism in the gas phase of the related system, 4-hydroxy-2-methyiminopentanoic acid, is 74 kJ mol(-)(1).

Clavanin bacterial sepsis control using a novel methacrylate nanocarrier.

Controlling human pathogenic bacteria is a worldwide problem due to increasing bacterial resistance. This has prompted a number of studies investigating peptides isolated from marine animals as a possible alternative for control of human pathogen infections. Clavanins are antimicrobial peptides isolated from the marine tunicate Styela clava, showing 23 amino acid residues in length, cationic properties, and also high bactericidal activity. In spite of clear benefits from the use of peptides, currently 95% of peptide properties have limited pharmaceutical applicability, such as low solubility and short half-life in the circulatory system. Here, nanobiotechnology was used to encapsulate clavanin A in order to develop nanoantibiotics against bacterial sepsis. Clavanin was nanostructured using EUDRAGIT(®) L 100-55 and RS 30 D solution (3:1 w:w). Atomic force, scanning electron microscopy and dynamic light scattering showed nanoparticles ranging from 120 to 372 nm in diameter, with a zeta potential of -7.16 mV and a polydispersity index of 0.123. Encapsulation rate of 98% was assessed by reversed-phase chromatography. In vitro bioassays showed that the nanostructured clavanin was partially able to control development of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, nanostructures did not show hemolytic activity. In vivo sepsis bioassays were performed using C57BL6 mice strain inoculated with a polymicrobial suspension. Assays led to 100% survival rate under sub-lethal sepsis assays and 40% under lethal sepsis assays in the presence of nanoformulated clavanin A until the seventh day of the experiment. Data here reported indicated that nanostructured clavanin A form shows improved antimicrobial activity and has the potential to be used to treat polymicrobial infections.

Structural and functional characterization of a multifunctional alanine-rich peptide analogue from Pleuronectes americanus.

Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets.

Role of the triad N46, S106 and T107 and the surface charges in the determination of the acidic pH optimum of digestive lysozymes from Musca domestica.

Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106-T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of D50 (catalytic nucleophile), whereas those residues are replaced by V109-A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D-S106V-T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces.

A draft of the human septin interactome.

BACKGROUND: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. CONCLUSIONS/SIGNIFICANCE: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the "group rule", i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

The acidic domain of hnRNPQ (NSAP1) has structural similarity to Barstar and binds to Apobec1.

Apobec1 edits the ApoB mRNA by deaminating nucleotide C(6666), which results in a codon change from Glutamate to stop, and subsequent expression of a truncated protein. Apobec1 is regulated by ACF (Apobec1 complementation factor) and hnRNPQ, which contains an N-terminal "acidic domain" (AcD) of unknown function, three RNA recognition motifs, and an Arg/Gly-rich region. Here, we modeled the structure of AcD using the bacterial protein Barstar as a template. Furthermore, we demonstrated by in vitro pull-down assays that 6xHis-AcD alone is able to interact with GST-Apobec1. Finally, we performed in silico phosphorylation of AcD and molecular dynamics studies, which indicate conformational changes in the phosphorylated form. The results of the latter studies were confirmed by in vitro phosphorylation of 6xHis-AcD by protein kinase C, mass spectrometry, and spectroscopic analyses. Our data suggest hnRNPQ interactions via its AcD with Apobec1 and that this interaction is regulated by the AcD phosphorylation.

The structural molecular biology network of the State of São Paulo, Brazil.

This article describes the achievements of the Structural Molecular Biology Network (SMolBNet), a collaborative program of structural molecular biology, centered in the State of São Paulo, Brazil, and supported by São Paulo State Funding Agency (FAPESP). It gathers twenty scientific groups and is coordinated by the scientific staff of the Center of Structural Molecular Biology, at the National Laboratory of Synchrotron Light (LNLS), in Campinas. The SMolBNet program has been aimed at 1) solving the structure of proteins of interest related to the research projects of the groups. In some cases, the choice has been to select proteins of unknown function or of possible novel structure obtained from the sequenced genomes of the FAPESP genomic program; 2) providing the groups with training in all the steps of the protein structure determination: gene cloning, protein expression, protein purification, protein crystallization and structure determination. Having begun in 2001, the program has been successful in both aims. Here, four groups reveal their participation in the program and describe the structural aspects of the proteins they have selected to study.

pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds.

The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.

Mechanism of action and NAD+-binding mode revealed by the crystal structure of L-histidinol dehydrogenase.

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to l-histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway. l-histidinol dehydrogenase (HisD, EC ) catalyzes the last two steps in the biosynthesis of l-histidine: sequential NAD-dependent oxidations of l-histidinol to l-histidinaldehyde and then to l-histidine. HisD functions as a homodimer and requires the presence of one Zn(2+) cation per monomer. We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, Zn(2+), and NAD(+) (best resolution is 1.7 A). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. Zn(2+) plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. NAD(+) binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.

Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa.

The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.

The structural molecular biology network of the State of São Paulo, Brazil

Esse artigo descreve realizações do Programa SMolBNet (Rede de Biologia Molecular Estrutural) do Estado de São Paulo, apoiado pela FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo). Ele reúne vinte grupos de pesquisa e é coordenado pelos pesquisadores do Laboratório Nacional de Luz Síncrotron (LNLS), em Campinas. O Programa SMolBNet tem como metas: Elucidar a estrutura tridimensional de proteínas de interesse aos grupos de pesquisa componentes do Programa; Prover os grupos com treinamento em todas as etapas de determinação de estrutura: clonagem gênica, expressão de proteínas, purificação de proteínas, cristalização de proteínas e elucidação de suas estruturas. Tendo começado em 2001, o Programa alcançou sucesso em ambas as metas. Neste artigo, quatro dos grupos descrevem suas participações, e discutem aspectos estruturais das proteínas que eles selecionaram para estudos.