Nucleotide Binding Assays Research Articles

Fluorescent, non-hydrolyzable nucleotide probes for ATP-activated ion channels.

In addition to its role as a ubiquitous, intracellular energy source and signaling molecule, adenosine 5’-triphosphate (ATP) also impacts extracellular signaling as both a neurotransmitter and a product of tissue damage. We previously developed a real-time nucleotide binding assay to study the gating of ATP-sensitive K+ (KATP) channels. Nucleotide binding was measured as Förster resonance energy transfer (FRET) between fluorescent, trinitrophenyl nucleotide derivatives and KATP channels tagged with the fluorescent, non-canonical amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP). In order to further probe the shape and chemistry of the nucleotide binding sites of ATP activated channels, we have synthesized and purified trinitrophenyl analogs of the weakly hydrolyzable adenosine-5'-(γ-thio)-triphosphate (ATP- γ-S) and the non-hydrolyzable nucleotides adenosine-5'-[(β,γ)-methyleno]triphosphate (AMP-PCP) and adenosine-5'-[(β,γ)-imido]triphosphate. The trinitrophenyl group was added to the ribose moiety of these nucleotides through an aqueous reaction with 2,4,6-trinitrobenzenesulfonic acid under basic conditions. The reaction products were purified by precipitation in ethanol followed by reversed-phase high-performance liquid chromatography. Products were verified using 1H and 31P nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and UV/visible absorbance. These compounds will be useful for exploring the requirement (if any) for ATP hydrolysis to activate KATP channels and the unique chemistry of the ATP binding sites of purinergic P2X-family receptors.

Characterzing the Auxiliary Nucleotide Binding Sites in RecBCD DNA Helicase, Towards Deciphering their Functional Role

RecBCD is a DNA helicase-nuclease, powering the initiation of ds break repair. It is a highly processive fast helicase, with unwinding rates approaching 1,600 bp/s. By employing a combination of equilibrium and time-resolved unwinding and binding experiments(both ensemble and single-molecule) to unravel RecBCD's molecular mechanism, we had demonstrated the existence of auxiliary binding sites in RecBCD, specifically in the RecC subunit, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. According to our model, RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites. The number of nucleotide-binding sites determined by equilibrium dialysis has been estimated to be at least four. While RecB and RecD, each have one known structurally and chemically well-defined nucleotide-binding site, the additional two (or more) auxiliary nucleotide-binding sites have not been located. Our current study strongly supports that they are likely to be located in RecC. In this work, we have attempted to abolish the nucleotide-binding sites in RecC by using a multi-step approach. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking, mass spectrometry and molecular docking studies. Based on this, several mutations in RecC were carefully designed and successfully cloned and purified to produce RecBCmutD. RecBCmutD has an activated DNA ATPase activity, with a higher KM,ATP. Furthermore, equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same biphasic binding isotherm as the WT RecBCD. Based on this, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Investigating the Role of the Auxiliary Nucleotide Binding Sites in the Recbcd DNA Helicase

RecBCD is highly processive and very fast DNA helicase-nuclease, responsible for the initiation of double-stranded break repair in E. coli. By employing a combination of equilibrium and time-resolved unwinding and binding experiments, both ensemble and single-molecule, we previously demonstrated the existence of auxiliary binding sites in RecBCD, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. We showed that RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites located in RecB and RecD subunits. Equilibrium dialysis indicated the number of nucleotide-binding sites to be at least four, and their likely location in the RecC subunit. In this current work, we provide further support for the functional role of the auxiliary sites, by abolishing the nucleotide-binding sites in RecC. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking and mass spectrometry, as well as molecular docking studies. Based on these predictions, several mutations in RecC were carefully designed, producing unique RecBCmutD. We have successfully cloned and purified RecBCmutD and shown that RecBCmutD has DNA-activated ATPase activity. Furthermore, an equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same binding isotherm as the WT RecBCD. Based on these observations, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A–E (TBCA–E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.

Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

A matter of energy stress: p38β meets mTORC1

A matter of energy stress: p38β meets mTORC1

Loss-of-Function Mutations in RAB18 Cause Warburg Micro Syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

Molecular basis of a novel oncogenic mutation in GNAO1

Heterotrimeric G proteins are molecular switches that control signal transduction, and their dysregulation can promote oncogenesis. Somatic mutations in GNAS, GNAI2 and GNAQ genes induce oncogenesis by rendering Gα subunits constitutively activated. Recently the first somatic mutation, arginine(243) → histidine (R243H) in the GNAO1 (Gαo) gene was identified in breast carcinomas and shown to promote oncogenic transformation when introduced into cells. Here, we provide the molecular basis for the oncogenic properties of the Gαo R243H mutant. Using limited proteolysis assays, nucleotide-binding assays, and single-turnover and steady-state GTPase assays, we demonstrate that the oncogenic R234H mutation renders Gαo constitutively active by accelerating the rate of nucleotide exchange; however, this mutation does not affect Gαo's ability to become deactivated by GTPase-activating proteins (GAPs) or by its intrinsic GTPase activity. This mechanism differs from that of previously reported oncogenic mutations that impair GTPase activity and GAP sensitivity without affecting nucleotide exchange. The constitutively active Gαo R243H mutant also enhances Src-STAT3 signaling in NIH-3T3 cells, a pathway previously shown to be directly triggered by active Gαo proteins to promote cellular transformation. Based on structural analyses, we propose that the enhanced rate of nucleotide exchange in Gαo R243H results from loss of the highly conserved electrostatic interaction of R243 with E43, located in the in the P-loop that represents the binding site for the α- and β-phosphates of the nucleotide. We conclude that the novel R234H mutation imparts oncogenic properties to Gαo by accelerating nucleotide exchange and rendering it constitutively active, thereby enhancing signaling pathways, for example, src-STAT3, responsible for neoplastic transformation.

CXCR2 Inverse Agonism Detected by Arrestin Redistribution

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

High-Throughput Screening for Small-Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

An Intramolecular Route for Coupling ATPase Activity in AAA+ Proteins for Transcription Activation

AAA+ proteins (ATPases associated with various cellular activities) contribute to many cellular processes and typically function as higher order oligomers permitting the coordination of nucleotide hydrolysis for functional output, which leads to substrate remodeling. The precise mechanisms that enable the relay of nucleotide hydrolysis to their specific functional outputs are largely unknown. Here we use PspF, a specialized AAA+ protein required for enhancer-dependent transcription activation in Escherichia coli, as a model system to address this question. We demonstrate that a conserved asparagine is involved in internal organization of the oligomeric ring, regulation of ATPase activity by "trans" factors, and optimizing substrate remodeling. We provide evidence that the spatial relationship between the asparagine residue and the Walker B motif is one key element in the conformational signaling pathway that leads to substrate remodeling. Such functional organization most likely applies to other AAA+ proteins, including Ltag (simian virus 40), Rep40 (Adeno-associated virus-2), and p97 (Mus musculus) in which the asparagine to Walker B motif relationship is conserved.

Structural and Functional Fingerprint of the Mitochondrial ATP-binding Cassette Transporter Mdl1 from Saccharomyces cerevisiae

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.

Nucleotide sequence and molecular characterization of a gene encoding GTP-binding protein from Streptococcus gordonii.

A 1286-bp fragment of chromosomal DNA from Streptococcus gordonii strain Challis was cloned and sequenced. The gene sgg consisted of 897-bp nucleotides encoding a 299-amino acid polypeptide (33,200 Da). The deduced amino acid sequence exhibited significant similarity to Era, G protein of Escherichia coli. The nucleotide binding assay demonstrated that recombinant Sgg bound [32P]GTP but not [32P]ATP, [32P]CTP, or [32P]UTP. These findings indicate that Sgg is a member of the G protein superfamily in the genus Streptococcus.

Gα selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN

GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Gα i/o–Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Gα o- and Gα i-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Gα i1, Gα i2, and Gα i3 and Gα o. Each of the four GPSM2 GoLoco motifs (36–43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST–GoLoco motifs to Gα i1-, Gα o-, and Gα s-subunits was assessed by surface plasmon resonance; all of the motifs bound Gα i1, but exhibited low affinity towards Gα o. GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Gα i1, Gα i2, and Gα i3. Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Gα o was significantly lower than that toward Gα i1, suggesting that the in vivo targets of GPSM2 are most likely to be Gα i-subunits.

Nucleotide binding to CARD12 and its role in CARD12-mediated caspase-1 activation

CARD12 (Ipaf/Clan) is an important regulator of caspase-1 activation. It belongs to the family of the nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins. The NBS domain of the NBS-LRR proteins contains putative ATP/GTPase-specific P-loop and Mg 2+-binding site motifs. However, the nucleotide-binding properties and the function of the NBS domain are unknown. We developed a nucleotide-binding assay and investigated nucleotide binding to CARD12. We find that the NBS domain of CARD12 contains a nucleotide-binding pocket with specificity for ATP/dATP. A point mutation in the P-loop (K175R) of the NBS domain abolishes ATP/dATP binding. We further demonstrate that the nucleotide-binding site is required for CARD12-mediated caspase-1 activation. CARD12 self-association and association with procaspase-1 in transfected cells were markedly decreased by the P-loop mutation K175R. Furthermore, the P-loop mutation greatly reduced caspase-1 activation-dependent proIL-1β processing. Thus, CARD12 function is dependent on the nucleotide-binding site. Our data provide insights into the molecular mechanisms of CARD12-mediated caspase-1 activation.

Nucleotide-dependent Formation of Catalytically Competent Dimers from Engineered Monomeric Ribonucleotide Reductase Protein R1

Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (alpha(2)) and R2 (beta(2)), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

Importance of Conserved N-domain Residues Thr441, Glu442, Lys515, Arg560, and Leu562 of Sarcoplasmic Reticulum Ca2+-ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.

A Cold-regulated Nucleic Acid-binding Protein of Winter Wheat Shares a Domain with Bacterial Cold Shock Proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.

Cytochrome c Promotes Caspase-9 Activation by Inducing Nucleotide Binding to Apaf-1

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.

Nucleotide sequence and molecular characterization of a gene encoding GTP-binding protein from Streptococcus gordonii

A 1286-bp fragment of chromosomal DNA from Streptococcus gordonii strain Challis was cloned and sequenced. The gene sgg consisted of 897-bp nucleotides encoding a 299-amino acid polypeptide (33 200 Da). The deduced amino acid sequence exhibited significant similarity to Era, G protein of Escherichia coli. The nucleotide binding assay demonstrated that recombinant Sgg bound [ 32P]GTP but not [ 32P]ATP, [ 32P]CTP, or [ 32P]UTP. These findings indicate that Sgg is a member of the G protein superfamily in the genus Streptococcus.