Interaction of substrate and effector binding sites in the ArsA ATPase

The ars operon of plasmid R773 confers resistance to antimonials and arsenicals in Escherichia coli by encoding an ATP-dependent extrusion system for the oxyanions. The catalytic subunit, the ArsA protein, is an ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates. Previous measurements suggested that the intrinsic fluorescence of tryptophans might be useful for examining binding of Mg2+ ATP and antimonite. In the present study an increase in intrinsic tryptophan fluorescence was observed upon addition of Mg2+ ATP. This enhancement was reversed by addition of antimonite. The ArsA protein contains four tryptophan residues: Trp159, Trp253, Trp522, and Trp524. The first two were altered to tyrosine residues by site-directed mutagenesis. Cells expressing both the arsAW159Y and arsAW253Y mutations retained resistance to arsenite, and the purified W159Y and W253Y proteins retained ATPase activity. While the intrinsic tryptophan fluorescence of the W253Y protein responded to addition of Mg2+ ATP, intrinsic tryptophan fluorescence in the purified W159Y protein was no longer enhanced by substrate. These results suggest that Trp159 is conformationally coupled to one or both of the nucleotide binding sites and provides a useful probe for the interaction of effector and substrate binding sites.     

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.     

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.     

Cooperative binding of ATP and MgADP in the sulfonylurea receptor is modulated by glibenclamide

The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.     

The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP

The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.     

Role of the J-domain in the cooperation of Hsp40 with Hsp70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.     

Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1

The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is initially rapid and decreases within about 1 min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of MgATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the enzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme in excess of 1, the initial ATPase activity was low and reached the steady state after about 30 s. Free Mg2+ in the assay mix also inhibited steady-state ATP hydrolysis by all forms of the enzyme. The results are consistent with a model in which two Mg2+ bind cooperatively, probably to the dissociable nucleotide-binding sites on CF1-epsilon. Thus, four different nucleotide-binding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the site to be involved in catalysis.     

Site-selected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) synthase is a bifunctional protein consisting of an NH2-terminal APS kinase and a COOH-terminal ATP sulfurylase. Both catalytic activities require ATP; the APS kinase domain involves cleavage of the beta-gamma phosphodiester bond of ATP, whereas the ATP sulfurylase domain involves cleavage of the alpha-beta phosphodiester bond of ATP. Previous mutational studies have suggested that beta-gamma phosphodiesterase activity involves a highly conserved NTP-binding P-loop motif located in the adenosine-5'-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in the ATP sulfurylase domain of PAPS synthases, a motif implicated in the alpha-beta phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP sulfurylase domain of human PAPS synthase (amino acids 425-428) was performed to examine this possibility. Either H425A or H428A mutation produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an important role for the HXGH histidines in the ATP sulfurylase activity of bifunctional PAPS synthase and support the hypothesis that the highly conserved HXGH motif found in the ATP sulfurylase domain of PAPS synthases is involved in ATP binding and alpha-beta phosphodiesterase activity.     

MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.     

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.     

Influence of substrates and MgADP on the time-resolved intrinsic fluorescence of phosphofructokinase from Escherichia coli. Correlation of tryptophan dynamics to coupling entropy

The influence of that MgADP and the substrate ligands MgATP and fructose 6-phosphate (Fru-6-P) have on the structure of E. coli phosphofructokinase (PFK) in the vicinity of the single tryptophan that exists in each subunit has been examined by employing both steady-state and time-resolved measurements of the tryptophan fluorescence. The accessibility of the tryptophan to iodide quenching is over 1 order of magnitude less than experienced by N-acetyltryptophanamide in solution but varies nonetheless with the state of ligation. Most, but not all, of these changes correlate with changes in the degree of local motion available to the tryptophan side chain as determined by steady-state and time-resolved polarization measurements. When the data obtained from differential polarization experiments are fit to a model in which the motion of the tryptophan side chain is able to move with high frequency within a cone of limited amplitude as part of an otherwise slowly tumbling spherical protein, it was found that ligands primarily affect the amplitude of the available local motion. By interpreting these effects with reference to the disproportionation equilibria which define the negative coupling free energy between MgADP and Fru-6-P and the positive coupling free energy between MgADP and MgATP, it is apparent that changes in the local motion amplitudes correlate with the sign of the component coupling entropy previously determined from van't Hoff analyses (Johnson & Reinhart, 1994).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase

Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIP1 encodes 1108 amino acids, including a hydrophobic membrane-associated domain in the NH2-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus cAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carboxyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and exhibited cAMP PDE activity which was inhibited by cilostamide, a specific cGI PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-L1 adipocytes. The deduced sequence of the second partial cDNA clone (RcGIP2 clone 53B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-L1 fibroblasts or adipocytes. We suggest that RcGIP1 represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.     

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.     

Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart

Sarcomere relaxation depends on dissociation of actin and myosin, which is regulated by a number of factors, including intracellular [MgATP] as well as MgATP hydrolysis products [MgADP] and inorganic phosphate [Pi], pHi, and cytosolic calcium concentration ([Ca2+]c). To distinguish the contribution of MgADP from the other regulators in the development of diastolic dysfunction, we used a strategy to increase free [MgADP] without changing [MgATP], [Pi], or pHi. This was achieved by applying a low dose of iodoacetamide to selectively inhibit the creatine kinase activity in isolated perfused rat hearts. [MgATP], [MgADP], [Pi], and [H+] were determined using 31P NMR spectroscopy. The [Ca2+]c and the glycolytic rate were also measured. We observed an approximately threefold increase in left ventricular end diastolic pressure (LVEDP) and 38% increase in the time constant of pressure decay (P < 0.05) in these hearts, indicating a significant impairment of diastolic function. The increase in LVEDP was closely related to the increase in free [MgADP]. Rate of glycolysis was not changed, and [Ca2+]c increased by 16%, which cannot explain the severity of diastolic dysfunction. Thus, our data indicate that MgADP contributes significantly to diastolic dysfunction, possibly by slowing the rate of cross-bridge cycling.     

Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.     

Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-

The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change. This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme. Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30%. The saturation curve for this compound is hyperbolic with a Kd of 6 microM. The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum. The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2. The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism. Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88. However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8%. Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex. The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes. The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2. These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E. coli, responsible for the inhibition of the enzyme activity by this ligand.     

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.     

Mapping of a defined neurocan binding site to distinct domains of tenascin-C

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.     

Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning

In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes. The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The NH2-terminal and COOH-terminal domains face the cytoplasm, separated by two membrane spanning segments and a short lumenal loop. The first cleavage occurs at Site-1 in the lumenal loop. The NH2-terminal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel cysteine panning method to identify the second cleavage site (Site-2) in human SREBP-2 as the Leu484-Cys485 bond that lies at the junction between the cytoplasmic NH2-terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH2-terminal and COOH-terminal sides of cysteine 485. The NH2-terminal fragments were tested for susceptibility to modification with Nalpha-(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls. Cysteines to the NH2-terminal side of cysteine 485 were retained on the NH2-terminal fragment, but cysteines to the COOH-terminal side of leucine 484 were lost. Leucine 484 is three residues to the COOH-terminal side of the tetrapeptide Asp-Arg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.     

The rate of CD4 decline as a determinant of progression to AIDS independent of the most recent CD4 count. The Italian Seroconversion Study

Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 buried in the interface between the subunits of the dimer. We studied the contributions of the tryptophans to fluorescence and catalytic dynamics by substituting Trp-314 with a leucine residue and making two compensatory mutations that were required to obtain a stable protein, leading to the triple mutant M303F-L308I-W314L enzyme. The substitutions increased by two- to sixfold the turnover numbers for ethanol oxidation, acetaldehyde reduction, and the dissociation constants of the coenzymes. The rate of the exponential burst phase for the transient oxidation of ethanol increased slightly, but the rate of dissociation of the enzyme-NADH complex still limited turnover of ethanol, as for wild-type enzyme. The three substitutions at the dimer interface apparently activate the enzyme by allowing more rapid conformational changes that accompany coenzyme binding, probably due to movement of the loop containing residues 293 to 298. The emission spectrum of M303F-L308I-W314L enzyme, which contains Trp-15, was redshifted compared to wild-type enzyme. Time-resolved fluorescence measurements with the triple mutant show that the decay of Trp-15 is dominated by a approximately 7-ns component. In the mutant enzyme with Trp-15 substituted with phenylalanine, the decay of Trp-314 is dominated by a approximately 4-ns component. Solute quenching data for wild-type enzyme and the mutants show that only Trp-15 is exposed to iodide and acrylamide, whereas Trp-314 is inaccessible. The luminescence properties of the tryptophan residues in the mutated enzymes are consistent with conclusions from studies of the wild-type enzyme [M. R. Eftink, 1992, Adv. Biophys. Chem. 2, 81-114].