Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F1 from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Identification of conventional and novel endothelin receptors in sheep choroid plexus cells

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent K_d's on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ET_A subtype with a K_d = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃) in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP₃, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ET_A subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide

Dimethylsulfoxide [Me₂SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F₁-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F₁ with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me₂SO, F₁ bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [³H]ADP to these sites was shown. A change in the conformation of F₁ in 30% (v/v) Me₂SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.     

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.     

Different classes of nucleotide binding sites in the (Na+ + K+)-ATPase studied by affinity labeling and nucleotide-dependent SH-group modifications

The ATP analog 6-[(3-carboxy-4-nitrophenyl)thiol]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) is slowly hydrolyzed at pH 7.4 by the (Na+ + K+)-ATPase, whereas it binds covalently at pH 8.5 and inhibits the enzyme irreversibly. Time courses of irreversible inhibition could only be fitted to a model in which the enzyme can exist in two slowly interchangeable states, one of which is enzymatically active and binds Nbs6ITP first reversibly and then covalently. Arguments that the covalent binding occurs at a low affinity nucleotide binding site are: (a) similarity of the Ki Nbs6ITP for the reversible and the irreversible inhibition and of K0.5 for ATP protection; (b) stoichiometry of covalent Nbs6ITP binding per alpha subunit of 0.8; and (c) change of complex substrate dependence of the enzyme to a Michaelis-Menten type after Nbs6ITP modification. This change in kinetics and the finding that the Nbs6ITP inactivation at a low affinity nucleotide binding site is increased by micromolar ADP concentrations indicates that the (Na+ + K+)-ATPase contains two different nucleotide binding sites. Since studies of nucleotide effects on enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) did not confirm the hypothesis of an SH-group in a nucleotide binding site, Nbs6ITP may bind to another functional group, e.g. to an OH-group of tyrosine.     

Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.     

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).     

Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

Characterization of an Asymmetric Occluded State of P-glycoprotein with Two Bound Nucleotides: IMPLICATIONS FOR CATALYSIS*

P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5′-(γ-thio)triphosphate (ATPγS), one of low affinity (K_d 0.74 mm), and one “occluded” nucleotide of 120-fold higher affinity (K_d 6 μm). ATPγS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPγS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPγS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[³H]phenylalanine (fML[³H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (K_d = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (K_d = 700 ± 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (K_d = 6.4 ± 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.     

Ligand Binding Properties of Bovine Liver Adenosine Kinase

Abstract

Bovine liver adenosine kinase displays a characteristic intrinsic fluorescence due to 3 tryptophans/mol. This fluorescence is very sensitive to ligand binding and was used to characterize the ligand binding sites of the enzyme. ADP or ATP showed a monophasic saturation curve consistent with the existence of one binding site. In contrast, adenosine and AMP gave biphasic saturation curves suggesting the existence of at least two binding sites with high and low affinity. These binding sites were further characterized by studying the complexation of adenosine kinase with O-(N-methylanthraniloyl)adenosine nucleoside or nucleotide analogues.     

Heterogeneity of Cortical and Hippocampal 5-HT1A Receptors: A Reappraisal of Homogenate Binding with 8-[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT_1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [³H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT_1A receptors (K_H= 0.7–0.8 nM) and lowaffinity (K_L= 22–36 nM) binding sites. The kinetics of [³H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [³H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT_1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT_1AHIGH and 5-HT_1A^LOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT_1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [³H]8-OH-DPAT to the 5-HT_1A^HIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT_1A^LOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT_1A^HIGH site corresponds to the classic 5-HT_1A receptor, whereas the novel 5-HT_1A^LOW binding site, labeled by 1 nM [³H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT_1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT_1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [³H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals.     

The Escherichia coli PriA helicase has two nucleotide-binding sites differing dramatically in their affinities for nucleotide cofactors. 1. Intrinsic affinities, cooperativities, and base specificity of nucleotide cofactor binding

Interactions of the Escherichia coli PriA helicase with nucleotide cofactors have been studied using the fluorescence titration and analytical ultracentrifugation techniques. Binding of unmodified cofactors was characterized by the fluorescence competition titration method. The obtained data establish that at saturation the PriA helicase binds two nucleotide molecules per protein monomer. This result corroborates with the primary structure of the protein, which contains sequence motifs implicated as putative nucleotide-binding sites. The intrinsic affinities of the binding sites differ by 2-4 orders of magnitude. Thus, the PriA helicase has a strong and a weak nucleotide-binding site. The binding sites differ dramatically in their properties. The strong site is highly specific for adenosine cofactors, while the weak site shows very modest base specificity. The affinities of the strong and weak binding sites for ATP are lower than the affinities for ADP, although both sites have similar affinity for the inorganic phosphate group. Unlike the weak site, the affinity of the strong site profoundly depends on the structure of the phosphate group of the ATP cofactor. Binding of unmodified nucleotides indicates the presence of positive cooperative interactions between bound cofactors (i.e., the existence of communication between the two sites). Magnesium cations are specifically involved in controlling the cofactor affinity for the strong site, while the affinity of the weak site is predominantly determined by interactions between the phosphate group and ribose regions of the cofactor and the protein matrix. The significance of these results for the activities of the PriA helicase is discussed.