bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Universal Role for Inositol 1,4,5-Trisphosphate-Mediated Signaling in Plant Gravitropism
Authors: Perera Imara Y. , Hung Chiu-Yueh , Brady Shari , Muday Gloria K. , Boss Wendy F. 
Published: December 2005

Abstract
Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants.
ArabidopsisACCELERATED CELL DEATH2 Modulates Programmed Cell Death
Authors: Yao Nan , Greenberg Jean T. 
Published: December 2005

AbstractThe Arabidopsis thaliana chloroplast protein ACCELERATED CELL DEATH2 (ACD2) modulates the amount of programmed cell death (PCD) triggered by Pseudomonas syringae and protoporphyrin IX (PPIX) treatment. In vitro, ACD2 can reduce red chlorophyll catabolite, a chlorophyll derivative. We find that ACD2 shields root protoplasts that lack chlorophyll from light- and PPIX-induced PCD. Thus, chlorophyll catabolism is not obligatory for ACD2 anti-PCD function. Upon P. syringae infection, ACD2 levels and localization change in cells undergoing PCD and in their close neighbors. Thus, ACD2 shifts from being largely in chloroplasts to partitioning to chloroplasts, mitochondria, and, to a small extent, cytosol. ACD2 protects cells from PCD that requires the early mitochondrial oxidative burst. Later, the chloroplasts of dying cells generate NO, which only slightly affects cell viability. Finally, the mitochondria in dying cells have dramatically altered movements and cellular distribution. Overproduction of both ACD2 (localized to mitochondria and chloroplasts) and ascorbate peroxidase (localized to chloroplasts) greatly reduces P. syringae–induced PCD, suggesting a pro-PCD role for mitochondrial and chloroplast events. During infection, ACD2 may bind to and/or reduce PCD-inducing porphyrin-related molecules in mitochondria and possibly chloroplasts that generate reactive oxygen species, cause altered organelle behavior, and activate a cascade of PCD-inducing events.
Elicitor-Mediated Oligomerization of the Tobacco N Disease Resistance Protein
Authors: Mestre Pere , Baulcombe David C. 
Published: December 2005

AbstractPlant nucleotide binding site–leucine-rich repeat (NBS-LRR) proteins are similar to the nucleotide binding oligomerization domain (NOD) protein family in their domain structure. It has been suggested that most NOD proteins rely on ligand-mediated oligomerization for function, and we have tested this possibility with the N protein of tobacco (Nicotiana tabacum). The N gene for resistance to Tobacco mosaic virus (TMV) is a member of the Toll-interleukin receptor (TIR)-NBS-LRR class of plant disease resistance (R) genes that recognizes the helicase domain from the TMV replicase. Using transient expression followed by immunoprecipitation, we show that the N protein oligomerizes in the presence of the elicitor. The oligomerization was not affected by silencing Nicotiana benthamiana ENHANCED DISEASE SUSCEPTIBILITY1 and N REQUIREMENT GENE1 cofactors of N-mediated resistance, but it was abolished by a mutation in the P-loop motif. However, loss-of-function mutations in the RNBS-A motif and in the TIR domain retain the ability to oligomerize. From these results, we conclude that oligomerization is an early event in the N-mediated resistance to TMV.
Competition between intercellular adhesion molecule‐1 and a small‐molecule antagonist for a common binding site on the αl subunit of lymphocyte function‐associated antigen‐1
Authors: Keating Susan M. , Clark Kevin R. , Stefanich Lisa D. , Arellano Fred , Edwards Caroline P. , Bodary Sarah C. , Spencer Steven A. , Gadek Thomas R. , Marsters James C. , Beresini Maureen H. 
Published: December 2005

AbstractThe lymphocyte function‐associated antigen‐1 (LFA‐1) binding of a unique class of small‐molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule‐1 (sICAM‐1) and A‐286982, which respectively define direct and allosteric competitive binding sites within LFA‐1's inserted (I) domain. All three molecules antagonized LFA‐1 binding to ICAM‐1‐Immunoglobulin G fusion (ICAM‐1‐Ig) in a competition ELISA, but only compound 3 and sICAM‐1 inhibited the binding of a fluorescein‐labeled analog of compound 3 to LFA‐1. Compound 3 and sICAM‐1 displayed classical direct competitive binding behavior with ICAM‐1. Their antagonism of LFA‐1 was surmountable by both ICAM‐1‐Ig and a fluorescein‐labeled compound 3 analog. The competition of both sICAM‐1 and compound 3 with ICAM‐1‐Ig for LFA‐1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross‐linking studies with a photoactivated analog of compound 3 localized the high‐affinity small‐molecule binding site to the N‐terminal 507 amino acid segment of the α chain of LFA‐1, a region that includes the I domain. In addition, cells transfected with a variant of LFA‐1 lacking this I domain showed no significant binding of a fluorescein‐labeled analog of compound 3 or ICAM‐1‐Ig. These results demonstrate that compound 3 inhibits the LFA‐1/ICAM‐1 binding interaction in a directly competitive manner by binding to a high‐affinity site on LFA‐1. This binding site overlaps with the ICAM‐1 binding site on the α subunit of LFA‐1, which has previously been localized to the I domain.
The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP‐induced rearrangement of the substrate binding domain
Authors: Moro Fernando , Fernández‐Sáiz Vanesa , Muga Arturo 
Published: December 2005

AbstractThe biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide‐induced cycling of DnaK between substrate‐accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide‐induced absorbance changes in the vibrational spectrum of wild‐type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C‐terminal α‐helical subdomain and another comprising only the N‐terminal ATPase domain), and a single‐point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time‐resolved infrared difference spectroscopy combined with the use of caged‐nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the α‐helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42°C).
High resolution structure of the HDGF PWWP domain: A potential DNA binding domain
Authors: Lukasik Stephen M. , Cierpicki Tomasz , Borloz Matthew , Grembecka Jolanta , Everett Allen , Bushweller John H. 
Published: December 2005

AbstractHepatoma Derived Growth Factor (HDGF) is an endogenous nuclear‐targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70–amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J‐couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C‐terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA‐binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA.
Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family
Authors: Svetlov Maxim S. , Kommer Aigar , Kolb Vyacheslav A. , Spirin Alexander S. 
Published: December 2005

AbstractMolecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell‐free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.
Finding biologically relevant protein domain interactions: Conserved binding mode analysis
Authors: Shoemaker Benjamin A. , Panchenko Anna R. , Bryant Stephen H. 
Published: December 2005

AbstractProteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well‐determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain interactions from the structural data. As a result, a set of conserved binding modes (CBMs) was created using the atomic detail of structure alignment data and the protein domain classification of the Conserved Domain Database. A conserved binding mode is inferred when different members of interacting domain families dock in the same way, such that their structural complexes superimpose well. Such domain interactions with recurring structural themes have greater significance to be biologically relevant, unlike spurious crystal packing interactions. Consequently, this study gives lower and upper bounds on the number of different types of interacting domain pairs in the structure database on the order of 1000–2000. We use CBMs to create domain interaction networks, which highlight functionally significant connections by avoiding many infrequent links between highly connected nodes. The CBMs also constitute a library of docking templates that may be used in molecular modeling to infer the characteristics of an unknown binding surface, just as conserved domains may be used to infer the structure of an unknown protein. The method's ability to sort through and classify large numbers of putative interacting domain pairs is demonstrated on the oligomeric interactions of globins.
Acid destabilization of the solution conformation of Bcl‐XL does not drive its pH‐dependent insertion into membranes
Authors: Thuduppathy Guruvasuthevan R. , Hill R. Blake 
Published: December 2005

AbstractRegulation of programmed cell death by Bcl‐xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH‐dependence similar to the translocation domain of diphtheria toxin, where an acid‐induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl‐xLΔTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near‐ and far‐UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl‐xLΔTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl‐xLΔTM (ΔG°) was determined to be 15.8 kcal·mol−1. Surprisingly, under conditions that favor a membrane conformation (pH 4.9), ΔG° was 14.6 kcal·mol−1. These results differ from those obtained with many other membrane‐insertable proteins where acid‐induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl‐xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl‐xL from intracellular proteases as it undergoes this conformational change essential for its activity.
Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure
Authors: Lee Seung‐Hyun , Carpenter John F. , Chang Byeong S. , Randolph Theodore W. , Kim Yong‐Sung 
Published: December 2005

AbstractHigh hydrostatic pressure (HHP)‐mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram‐negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox‐shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox‐shuffling agent resulted in solubilization yields of ∼42%–58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L‐arginine (0.5 M), Tween 20 (0.1 mM), or Triton X‐100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (∼70%–100%) to achieve refolding yields of ∼55%–78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.
pH driven conformational dynamics and dimer‐to‐monomer transition in DLC8
Authors: Mohan P.M. Krishna , Barve Maneesha , Chatterjee Amarnath , Hosur Ramakrishna V. 
Published: December 2005

AbstractDynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15N transverse relaxation measurements. At pH 7 and below until 5, partial protonation and consequent interconversion between molecules carrying protonated and neutral histidines, causes conformational dynamics in the dimeric protein and this increases with decreasing pH. Enhanced dynamics in turn leads to partial loosening of the structure. This would have implications for different efficacies of binding by target proteins due to small variations in pH in different parts of the cell, and hence for cargo trafficking from one part to another. Below pH 5, enhanced charge repulsions, partial loss of hydrophobic interactions, and destabilization of H‐bonds across the dimer interface cause further loosening of the dimeric structure, leading eventually to the dissociation of the dimer.
Plasmodium falciparum glutathione S‐transferase—Structural and mechanistic studies on ligand binding and enzyme inhibition
Authors: Hiller Nicole , Fritz‐Wolf Karin , Deponte Marcel , Wende Wolfgang , Zimmermann Herbert , Becker Katja 
Published: December 2005

AbstractGlutathione S‐transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high‐ and a low‐affinity binding site. This is supported by a clear uncompetitive inhibition type. Site‐directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH‐dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 Å resolution X‐ray structure of PfGST cocrystallized with the inhibitor S‐hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C‐terminal end of helix α4 and at the S‐hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.
Solution structure of the N‐terminal A domain of the human voltage‐gated Ca2+channel β4a subunit
Authors: Vendel Andrew C. , Rithner Christopher D. , Lyons Barbara A. , Horne William A. 
Published: December 2005

AbstractCa2+ channel β subunits regulate trafficking and gating (opening and closing) of voltage‐dependent Ca2+ channel α 1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A–E) that are related to the large family of membrane associated guanylate‐kinase (MAGUK) proteins. The crystal structures of the β subunit core, B–D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N‐terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel β subunits (β1–β4). Furthermore, this domain undergoes alternative splicing to create multiple N‐terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human β4a subunit, a splice variant that we have shown previously to have α 1 subunit subtype‐specific effects on Ca2+ channel trafficking and gating.
Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3
Authors: Arai Ryoichi , Kukimoto‐Niino Mutsuko , Kuroishi Chizu , Bessho Yoshitaka , Shirouzu Mikako , Yokoyama Shigeyuki 
Published: December 2005

AbstractPH0459, from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, is a probable haloacid dehalogenase with a molecular mass of 26,725 Da. Here, we report the 2.0 Å crystal structure of PH0459 (PDB ID: 1X42) determined by the multiwavelength anomalous dispersion method. The core domain has an α/β structure formed by a six‐stranded parallel β‐sheet flanked by six α‐helices and three 310‐helices. One disulfide bond, Cys186–Cys212, forms a bridge between an α‐helix and a 310‐helix, although PH0459 seems to be an intracellular protein. The subdomain inserted into the core domain has a four‐helix bundle structure. The crystal packing suggests that PH0459 exists as a monomer. A structural homology search revealed that PH0459 resembles the l‐2‐haloacid dehalogenases l‐DEX YL from Pseudomonas sp. YL and DhlB from Xanthobacter autotrophicus GJ10. A comparison of the active sites suggested that PH0459 probably has haloacid dehalogenase activity, but its substrate specificity may be different. In addition, the disulfide bond in PH0459 probably facilitates the structural stabilization of the neighboring region in the monomeric form, although the corresponding regions in l‐DEX YL and DhlB may be stabilized by dimerization. Since heat‐stable dehalogenases can be used for the detoxification of halogenated aliphatic compounds, PH0459 will be a useful target for biotechnological research.
Solution structure of the C4 zinc finger domain of HDM2
Authors: Yu Grace W. , Allen Mark D. , Andreeva Antonina , Fersht Alan R. , Bycroft Mark 
Published: December 2005

AbstractHDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 310 helix followed by four β‐strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.