Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with [alpha-32P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.     

Structure-function relationships in an anion-translocating ATPase

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.     

Complementation between nucleotide binding domains in an anion-translocating ATPase.

The catalytic component of the oxyanion-translocating ATPase of the plasmid-encoded ars operon of Escherichia coli is a homodimer of the ArsA protein. This enzyme is an oxyanion-stimulated ATPase with two consensus nucleotide binding sequences in each subunit, one in the N-terminal (A1) half and one in the C-terminal (A2) half of the ArsA protein. The two halves of both the arsA gene and the ArsA protein exhibit similar nucleotide and amino acid sequences, respectively. The two halves of the arsA gene were subcloned into compatible plasmids. Neither alone was sufficient to confer resistance, but cells in which the arsA1 and arsA2 half genes were coexpressed were resistant to arsenicals. Genetic complementation was also observed in cells bearing plasmids with point mutations in the two halves of the arsA gene and between cells with plasmids carrying combinations of the arsA1 or arsA2 subclones and point mutations. In every case, complementation was observed only when one plasmid contained a wild-type arsA1 sequence and the other contained a wild-type arsA2 sequence. These results demonstrate that both sites are required for resistance but that the two nucleotide binding domains need not reside in a single polypeptide. We propose a model in which the ArsA dimer has two catalytic units, each composed of an A1 domain from one monomer and an A2 domain from the other monomer.     

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.     

Substrate-induced dimerization of the ArsA protein, the catalytic component of an anion-translocating ATPase

The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite, suggesting that the binding of antimonite increased the affinity of ArsA protein for FSBA. Incorporation of [14C]FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.     

Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit.

The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.     

Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.     

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102–105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.     

The structure of the nucleotide-binding site of kinesin.

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.     

Probing the nucleotide-binding site of Escherichia coli succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246alpha) intermediate. Two potential nucleotide-binding sites were predicted in the beta-subunit, and have been differentiated by photoaffinity labeling with 8-N3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N3-ATP is a suitable analogue for probing the nucleotide-binding site of SCS. Two tryptic peptides from the N-terminal domain of the beta-subunit were labeled with 8-N3-ATP. These corresponded to residues 107-119beta and 121-146beta, two regions lying along one side of an ATP-grasp fold. A mutant protein with changes on the opposite side of the fold (G53betaV/R54betaE) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by succinyl-CoA and Pi. A mutant protein designed to probe nucleotide specificity (P20betaQ) had a Km(app) for GTP that was more than 5 times lower than that of wild-type SCS, whereas parameters for the other substrates remained unchanged. Mutations of residues in the C-terminal domain of the beta-subunit designed to distrupt one loop of the Rossmann fold (I322betaA, and R324betaN/D326betaA) had the greatest effect on the binding of succinate and CoA. They did not disrupt the phosphorylation of SCS with nucleotides. It was concluded that the nucleotide-binding site is located in the N-terminal domain of the beta-subunit. This implies that there are two active sites approximately 35 A apart, and that the H246alpha loop moves between them during catalysis.     

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.     

The non-catalytic nucleotide-binding site of mitochondrial ATPase is localised on the alpha-subunit(s) of factor F1

The incubation of isolated factor F1 with the di-aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9--1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 micro M. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the alpha-subunit(s) of factor F1. When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30 000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane-bound factor F1 covalently binds 0.2--0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the alpha subunit(s) of factor F1. The modification of membrane-bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non-catalytic site of mitochondrial ATP ase located on the alpha-subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane-bound ATPase.     

A model of the nucleotide-binding site in tubulin

Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.     

On the existence of a nucleotide-binding regulatory site on brain hexokinase.

Binding of ADP to rat brain hexokinase provided protection against inactivation of the enzyme by glutaraldehyde or by chymotryptic digestion. Graphical analysis of the inactivation experiments was, in both cases, consistent with the existence of a single ADP binding site and a K_d ≈ 3mM for the hexokinase-ADP complex. Both Cibacron Blue F3GA and tetraiodofluorescein, previously found to have a general affinity for nucleotide binding sites, were competitive (vs. ATP) inhibitors of the enzyme, suggesting that they bound only to the site occupied by the nucleotide substrate, ATP. While alternate interpretations cannot be excluded, it is felt that these results are most consistent with the view that there is a single nucleotide binding site on the enzyme. They thereby may serve to stimulate a search for alternative explanations for the complex inhibitory pattern of ADP which had previously been attributed to the existence of two ADP binding sites on the enzyme (J. Ning, D.L. Purich, and H.J. Fromm, $\text{J. Biol. Chem. 244}$, 3840–3846 (1969).     

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.     

The nucleotide-binding site of the Escherichia coli DnaC protein

The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3′-O-(N-methylantraniloyl)-5′-triphosphate (MANT-ATP) and 3′-O-(N-methylantraniloyl)-5′-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.     

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart.

Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.