Conserved glycine residues in the P-loop of ATP synthases form a doorframe for nucleotide entrance

The phosphate binding loop (GXXXXGKT(S)) is conserved in several mononucleotide-binding proteins with similar three-dimensional structures. Although variations in other amino acids have been noted, the first glycine and glycine-lysine residues are highly conserved in all enzymes, whose role is yet to be understood. Alanine substitutions for critically positioned glycines—G234, G237, and G239—were generated for the catalytic A-subunit of A-ATP synthase from Pyrococcus horikoshii OT3, and their crystal structures were determined. They showed altered conformation for the phosphate binding loop, with G234A and G237A becoming flat and with G239A taking an intermediate conformation, resulting in the active-site region being closed to nucleotide entry. Furthermore, the essential amino acids S238 and K240, which normally interact with the nucleotide, become inaccessible. These mutant structures demonstrate the role of the strictly conserved glycine residues in guarding the active-site region for nucleotide entrance in archaea-type ATP synthases.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.     

Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1 complex of the A1AO ATP synthase

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A₁A_O ATP synthases (A₃B₃CDE₂FG₂ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A₃B₃D- and A₃B₃DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.     

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk

A₁A_O ATP synthases are the major energy converters of archaea. They are composed of an A₁ region that synthesizes ATP and an integral part A_O that conducts ions. Subunit E is a component of the peripheral stalk that links the A₁ with the A_O part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases.     

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F₁ ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β_DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.     

Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

ATP synthases from archaea: The beauty of a molecular motor

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A₁A_O ATP synthases, a class of ATP synthases distinct from the F₁F_O ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V₁V_O ATPases of eukaryotes. A₁A_O ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A₁A_O ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A₁A_O ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H⁺, Na⁺ or even H⁺ and Na⁺ using enzymes. The evolution of the H⁺ binding site to a Na⁺ binding site and its implications for the energy metabolism and physiology of the cell are discussed.     

Substrate Binding and Catalysis in Carbamate Kinase Ascertained by Crystallographic and Site-Directed Mutagenesis Studies: Movements and Significance of a Unique Globular Subdomain of This Key Enzyme for Fermentative ATP Production in Bacteria

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 Å resolution and containing bound MgADP, and the other at 2.1 Å resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K_m^CP and k_cat triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.     

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V₀V₁) as a result of ADP inhibition occurs as part of the normal mechanism of V₀V₁ of Thermus thermophilus but not V₀V₁ of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V₁ consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V₁ is central to increasing binding affinity of the chimeric V₁ for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V₁ with E. hirae V₁ strongly suggest that the A subunit adopts a conformation in T. thermophilus V₁ different from that in E. hirae V₁. This key difference results in ADP inhibition of T. thermophilus V₁ by abolishing the binding affinity for phosphate during ATP hydrolysis.     

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

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.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.     

Origin of apparent negative cooperativity of F1-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F₁-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F₁-ATPase, α_(W463F)3β_(Y341W)3γ and α_{(K175A/T176A/W463F)3}β_(Y341W)3γ. For α_(W463F)3β_(Y341W)3γ, apparent K_m's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K_m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent K_m of ATPase activity (10 μM) roughly agreed with the highest K_m of fluorescence measurements (27 μM). The results indicate that in case of α_(W463F)3β_(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K_m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, the K_m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α_{(K175A/T176A/W463F)3}β_(Y341W)3γ was rather linear compared with that of α_(W463F)3β_(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F₁-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K_m's of 100-300 μM and 1-30 μM range for wild-type F₁-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A₃B₃ hexameric complex, it might not function as a bona fide ATP synthase.     

Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase Domains

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k_cat/K_m) of D1 ATP hydrolysis 280-fold, by increasing k_cat 7-fold and decreasing K_m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.     

ATP hydrolysis in ATP synthases can be differently coupled to proton transport and modulated by ADP and phosphate: A structure based model of the mechanism

In the ATP synthases of Escherichia coli ADP and phosphate exert an apparent regulatory role on the efficiency of proton transport coupled to the hydrolysis of ATP. Both molecules induce clearly biphasic effects on hydrolysis and proton transfer. At intermediate concentrations (～ 0.5–1 µM and higher) ADP inhibits hydrolysis and proton transfer; a quantitative analysis of the fluxes however proves that the coupling efficiency remains constant in this concentration range. On the other hand at nanomolar concentrations of ADP (a level obtainable only using an enzymatic ATP regenerating system) the efficiency of proton transport drops progressively, while the rate of hydrolysis remains high. Phosphate, at concentrations ≥ 0.1 mM, inhibits hydrolysis only if ADP is present at sufficiently high concentrations, keeping the coupling efficiency constant. At lower ADP levels phosphate is, however, necessary for an efficiently coupled catalytic cycle. We present a model for a catalytic cycle of ATP hydrolysis uncoupled from the transport of protons. The model is based on the available structures of bovine and yeast F₁ and on the known binding affinities for ADP and P_i of the catalytic sites in their different functional states. The binding site related to the inhibitory effects of P_i (in association with ADP) is identified as the α_HCβ_HC site, the pre-release site for the hydrolysis products. We suggest, moreover, that the high affinity site, associated with the operation of an efficient proton transport, could coincide with a conformational state intermediate between the α_TPβ_TP and the α_DPβ_DP (similar to the transition state of the hydrolysis/synthesis reaction) that does not strongly bind the ligands and can exchange them rather freely with the external medium. The emptying of this site can lead to an unproductive hydrolysis cycle that occurs without a net rotation of the central stalk and, consequently, does not translocate protons.     

Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3,N⁶-etheno-ATP. phosphate/carbamoyl-d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme.We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3,N⁶-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose.Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies.Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation.Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London &; Schmidt (1972). We suggest, however, a modification of the London &; Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.     

Role of the conserved lysine within the Walker A motif of human DMC1

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1_K132R) or alanine (hDMC1_K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1_K132R and hDMC1_K132A variants were devoid of ATP hydrolysis. The hDMC1_K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1_K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1_K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.