Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein

Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.     

Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors

Global conformational and oligomeric states of the Escherichia coli replicative factor DnaC protein in the absence and presence of magnesium and nucleotide cofactors, ATP and ADP, and their fluorescent analogues, MANT-ATP and MANT-ADP, have been examined using analytical sedimentation velocity and time-dependent fluorescence anisotropy techniques. In solution, the DnaC protein exists exclusively as a monomer over a large protein concentration range. The value of s(degrees) (20, w)= 2.45 +/- 0.07 S indicates that the protein molecule has an elongated shape. When modeled as a prolate ellipsoid of revolution, the hydrated DnaC protein has an axial ratio of 4.0 +/- 0.6 with long axis a = 112 A and the short axis b = 28 A, respectively. The presence of magnesium or nucleotide cofactors, ATP or ADP, does not affect the global conformation of the protein and its monomeric state. These data indicate that recently found cooperative interactions between the DnaC molecules, in the complex with the DnaB helicase, are induced by the binding to the helicase, i.e., they are not the intrinsic property of the DnaC protein. Fluorescence anisotropy decays of the DnaC-MANT-ATP and DnaC-MANT-ADP complexes indicate that the protein has a rigid global structure on the nanosecond time scale, little affected by the nucleotide cofactors. Nevertheless, the complex with ATP has a more flexible structure, while the complex with ADP is more rigid, with the protein molecule assuming a more elongated shape. Magnesium exerts control only on the complex with the ATP analogue. In the absence of magnesium, the ATP analogue is firmly held in the binding site. In the presence of Mg(2+), this fixed location is released and the analogue is allowed to assume a flexible conformational state. The significance of the results for the functioning of the DnaC protein is discussed.     

The E. coli replication factor DnaC protein exists in two conformations with different nucleotide binding capabilities. I. Determination of the binding mechanism using ATP and ADP fluorescent analogues

The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E. coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique. The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration. Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein. Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding. The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations, as defined by In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)(2) that binds the nucleotide. The lack of any cofactor binding to the (DnaC)(1) state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein. Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation. The (C)(1) intermediate dominates the distribution of the DnaC protein population in the presence of cofactors. The formation of (C)(1) is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors. Transition to (C)(2) places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity approximately 8-fold higher than that of the free cofactor. The significance of these results for the functioning of the DnaC protein is discussed.     

Kinetics of the E. coli replication factor DnaC protein-nucleotide interactions. II. Fluorescence anisotropy and transient,dynamic quenching stopped-flow studies of the reaction intermediates

The nature of the intermediates in the binding of MANT-ATP and MANT-ADP to the E. coli replicative factor DnaC protein (accompanying paper) has been examined using the fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow techniques. Using molar fluorescence intensities of individual intermediates of the reaction, we derived the Stern-Volmer equation that provides a direct method to quantitatively address the quenching of the fluorescence of a transient intermediate by an external, neutral quencher. The data indicate that in the first intermediate, (C)(1), the solvent has full access to the MANT group. Thus, the nucleotide-binding site is located on the surface of the protein, fully open to the solvent. Moreover, formation of the first intermediate does not affect the structure of the binding site. On the other hand, in the second intermediate, (C)(2), the entire binding site changes its conformation, resulting in diminished access of the solvent to the bound nucleotide. The time course of the fluorescence anisotropy in the reaction provides direct, unique insight into the mobility of bound nucleotides in each intermediate. The analysis is facilitated by the fact that the anisotropy can be expressed as a function of the relative molar intensities and steady-state anisotropies of the individual intermediates. The major decrease of the nucleotide mobility occurs in the formation of the first intermediate and reflects the fact that the MANT group is immobilized to a similar extent as the ribose region of the bound nucleotides. Transition to the second intermediate and closing of the binding site leads to only a moderate, additional decrease of nucleotide mobility. The temperature effect on the studied interactions indicates that the formation of individual intermediates is accompanied by very different enthalpy and entropy changes predominantly generated from the structural changes of the protein. Analysis of the salt effect indicates that the net release of a single ion, observed in equilibrium studies, occurs in the formation of the first intermediate. The lack of any salt effect on the (C)(1) <--> (C)(2) transition indicates that the closing of the binding site does not include a net ion release or uptake. Moreover, prior to the nucleotide binding, the conformational transition of the DnaC protein is exclusively controlled by the nucleotide binding and release.     

Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex

Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and fluorescence energy transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB-DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(+/-0.5)x10(5)M(-1) and cooperativity parameter sigma=21+/-5. These data suggest strongly that the DnaC-DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC-DnaB-ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC-DnaB-ssDNA has been determined using the fluorescence energy transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase-DnaC protein complex is discussed.     

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.     

The DnaC helicase loader is a dual ATP/ADP switch protein

Helicases are transferred to replication origins by helicase loading factors. The Escherichia coli DnaC and eukaryotic Cdc6/18 helicase loaders contain ATP sites and are both members of the AAA+ family. One might expect that ATP is required for helicase loading; however, this study on DnaC illustrates that ATP is not actually needed for DnaC to load helicase onto single-strand DNA (ssDNA). In fact, it seems to be a paradox that after transfer of helicase to DNA, DnaC-ATP inhibits helicase action. In addition, ATP is required for DnaC function at an early step in oriC replication in which ATP stimulates ssDNA binding by DnaC, leading to expansion of the ssDNA bubble at the origin. Two cofactors, ssDNA and DnaB, trigger hydrolysis of ATP, converting DnaC to the ADP form that no longer inhibits DnaB. These observations have led to the idea that DnaC is a 'dual' switch protein, where both the ATP and the ADP forms are sequentially required for replication. This dual switching process may underlie the sensitivity of DnaB to even small fluctuations in DnaC levels.     

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 Escherichia coli PriA Helicase-Double-Stranded DNA Complex: Location of the Strong DNA-Binding Subsite on the Helicase Domain of the Protein and the Affinity Control by the Two Nucleotide-Binding Sites of the Enzyme

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5 ± 1 bp, that is, dramatically lower than 20 ± 3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.     

Stability and dynamics of G-actin: back-door water diffusion and behavior of a subdomain 3/4 loop.

Molecular dynamics simulations have been performed on solvated G-actin bound to ADP and ATP, starting with the crystal structure of the actin-DNase 1 complex, including a Ca2+ or Mg2+ ion at the high-affinity divalent cation-binding site. Water molecules have been found to enter the nucleotide-binding site (phosphate vicinity) along two pathways, from the side where the nucleotide base is exposed to water, as well as from the opposite side. The water channels suggest a "back-door" mechanism for ATP hydrolysis in which the phosphate is released to a side opposite that of nucleotide binding and unbinding. The simulations also reveal a propensity of G-actin to alter its crystallographic structure toward the filamentous structure. Domain movement closes the nucleotide cleft, the movement being more pronounced for bound Mg2+. The conformational change is interpreted as a response of the system to missing water molecules in the crystal structure. The structures arising in the simulations, classified according to nucleotide cleft separation and radius of gyration of the protein, fall into two distinct clusters: a cluster of states that are similar to the G-actin crystal structure, and a cluster of states with small cleft separation and with the subdomain 3/4 loop 264-273 detached from the protein. The latter states resemble the putative filamentous structure of actin, in which the loop connects the two strands of the actin filament.     

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2′-P3′ region of the active site. In contrast, the inhibitor CGS27023A, which is t hought to bind in the P1-P1′ region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant ofK _i=22 μM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40±0.17 μM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.     

Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains

We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing the protein with the capacity to be allosterically regulated by adenine nucleotides (J?msen, J., Tuominen, H., Salminen, A., Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327–333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated by diadenosine polyphosphates (AP _n A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2′(3′)-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding. The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it to more rapidly undergo conformational changes in response to binding.     

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.     

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1

The pH-dependence of ADP and ATP affinity for CF₁ tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF₁ ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF₁ ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF₁ tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.Abbreviations CF₁, BF₁, and MF₁ coupling factors of chloroplasts, bacteria, and mitochondria, respectively - AdN adenine nucleotide     

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.     

Allostery in Hsp70 Chaperones Is Transduced by Subdomain Rotations

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's.It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD?Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.     

Toward an Adequate Scheme for the ATP Synthase Catalysis

The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and P_i but not ATP. At a closed site, tightly bound ADP and P_i are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120° rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and P_i are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The _B structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered.     

Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter

The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.     

Kinetic mechanisms of the nucleotide cofactor binding to the strong and weak nucleotide-binding site of the Escherichia coli PriA helicase. 2

Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.     

Molecular Interplay between the Replicative Helicase DnaC and Its Loader Protein DnaI from Geobacillus kaustophilus

Helicase loading factors are thought to transfer the hexameric ring-shaped helicases onto the replication fork during DNA replication. However, the mechanism of helicase transfer onto DNA remains unclear. In Bacillus subtilis, the protein DnaI, which belongs to the AAA+ family of ATPases, is responsible for delivering the hexameric helicase DnaC onto DNA. Here we investigated the interaction between DnaC and DnaI from Geobacillus kaustophilus HTA426 (GkDnaC and GkDnaI, respectively) and determined that GkDnaI forms a stable complex with GkDnaC with an apparent stoichiometry of GkDnaC₆-GkDnaI₆ in the absence of ATP. Surface plasmon resonance analysis indicated that GkDnaI facilitates loading of GkDnaC onto single-stranded DNA (ssDNA) and supports complex formation with ssDNA in the presence of ATP. Additionally, the GkDnaI C-terminal AAA+ domain alone could bind ssDNA, and binding was modulated by nucleotides. We also determined the crystal structure of the C-terminal AAA+ domain of GkDnaI in complex with ADP at 2.5 Å resolution. The structure not only delineates the binding of ADP in the expected Walker A and B motifs but also reveals a positively charged region that may be involved in ssDNA binding. These findings provide insight into the mechanism of replicative helicase loading onto ssDNA.