ATP binding,not hydrolysis,at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.     

Analysis of the human MutLalpha.MutSalpha complex

Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.     

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.     

Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.     

Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.     

Differential and simultaneous adenosine di- and triphosphate binding by MutS

The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood. As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS. Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent. Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein. Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range. These findings suggest that the protein exists primarily as the ATP.MutS.ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo.     

Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPgammaS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.     

DNA helicase RepA: cooperative ATPase activity and binding of nucleotides

The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.     

Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22 degrees C and 4 degrees C and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH2, all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis.

The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+). Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge. AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.     

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.     

The DNA binding properties of the Escherichia coli RecQ helicase

The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.     

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.     

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.     

Thermodynamics of nucleotide binding to NBS-I of the Bacillus subtilis preprotein translocase subunit SecA.

SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non-hydrolyzable ATPgammaS is enthalpy-driven (DeltaH(0) of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (DeltaS(0) of -18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (DeltaC(p)=-780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.     

Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.     

The role of the bound nucleotide in the polymerization of actin.

Three mucleotides, ATP, ADP, and an unsplit-table analog of ATP (adenylyl imidodiphosphate (AMPPNP)), were bound to monomeric actin, and their effects on the rate and extent of the actin polymerization were studied. The kinetics of polymerization, assayed by the change in OD232, followed a simple exponential curve. The rates of polymerization were equal for bound ATP and AMPPNP; both of which were three to five times faster than the rate for ADP. The concentration of actin monomers in apparent equilibrium with the polymer, G(180 degrees longitude), was determined. Values of G(180 degrees longitude) in 100 mM KCl were found for different nucleotides to be: G-ATP(180 degrees longitude) = 0.7 mu-M, G-AMPPNP(180 degrees longitude) = 0.8 MU-M, and G-ADP(180 degrees longitude) = 3.4 mu-M. The equilibrium constant of the polymerization is given by K = [G(180 degrees longitude)]-minus 1 when no nucleotide is split. The polymerization of actin-ATP is more complex due to the splitting of the nucleotide and our data require that this polymerization involves more than one step. The kinetic parameters for the polymerization of actin-ATP can be explained by a simple scheme in which the nucleotide dephosphorylation occurs in a step following the polymerization step. The conclusions are: (1) the binding of ATP to actin monomer promotes polymerization slightly more than the binding of ADP, (2) actin bound ATP provides less than 4 kJ/mol of free energy to promote polymerization, and (3) the dephosphorylation of the nucleotide is not coupled to polymerization.