Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin

Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal structure of human Srx (hSrx) exhibits a new protein fold and a unique nucleotide binding motif containing the Gly98-Cys99-His100-Arg101 sequence at the N-terminus of an alpha-helix. HPLC analysis of the reaction products has confirmed that the site of ATP cleavage is between the beta- and gamma-phosphate groups. Cys99 and the gamma-phosphate of ATP, modeled within the active site of the 2.0 A ADP product complex structure, are adjacent to large surface depressions containing additional conserved residues. These features and the necessity for significant remodeling of the Prx structure suggest that the interactions between hSrx and typical two-Cys Prxs are specific. Moreover, the concave shape of the hSrx active site surface appears to be ideally suited to interacting with the convex surface of the toroidal Prx decamer.     

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Protein Engineering of the Quaternary Sulfiredoxin��Peroxiredoxin Enzyme��Substrate Complex Reveals the Molecular Basis for Cysteine Sulfinic Acid Phosphorylation

Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO₂⁻. This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg²⁺-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme·substrate complex has been trapped. Here, we present the 2.1 Å crystal structure of human Srx in complex with PrxI, ATP, and Mg²⁺. The Cys⁵² sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe⁵⁰ in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp⁵² within 4.3 Å of the γ-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO₂⁻ is the first chemical step.     

Reduction of cysteine sulfinic acid in peroxiredoxin by sulfiredoxin proceeds directly through a sulfinic phosphoryl ester intermediate

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO(2)(-). This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the gamma-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8A resolution crystal structure of Srx in complex with ATP and Mg(2+). This structure reveals the role of the Mg(2+) ion to position the gamma-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO(2)(-) species, support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.     

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.     

Crystal structure of the thrombin mutant D221A/D222K: the Asp222:Arg187 ion-pair stabilizes the fast form

The thrombin mutant D221A/D222K (ARK) does not bind Na+ and has interesting functional properties intermediate between those of the slow and fast forms of wild type. We solved the X-ray crystal structure of ARK bound at exosite I with a fragment of hirudin at 2.4-A resolution. The structure shows a slight collapse of the 186 and 220 loops into the Na+ binding site due to disruption of the Asp222:Arg187 ion-pair. The backbone O atoms of Arg221a and Lys224 are shifted into conformations that eliminate optimal interaction with Na+. A paucity of solvent molecules in the Na+ binding site is also noted, by analogy to what is seen in the structure of the slow form. These findings reinforce the crucial role of the Asp222:Arg187 ion-pair in stabilizing the fast form of thrombin.     

Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp

The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA. The core polymerase is tethered to the template by beta, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta. However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10(5)-fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of beta. Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.     

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.     

Crystal structures of hydrogenase maturation protein HypE in the Apo and ATP-bound forms

The hydrogenase maturation protein HypE serves an essential function in the biosynthesis of the nitrile group, which is subsequently coordinated to Fe as CN(-) ligands in [Ni-Fe] hydrogenase. Here, we present the crystal structures of HypE from Desulfovibrio vulgaris Hildenborough in the presence and in the absence of ATP at a resolution of 2.0 A and 2.6 A, respectively. Comparison of the apo structure with the ATP-bound structure reveals that binding ATP causes an induced-fit movement of the N-terminal portion, but does not entail an overall structural change. The residue Cys341 at the C terminus, whose thiol group is supposed to be carbamoylated before the nitrile group synthesis, is completely buried within the protein and is located in the vicinity of the gamma-phosphate group of the bound ATP. This suggests that the catalytic reaction occurs in this configuration but that a conformational change is required for the carbamoylation of Cys341. A glutamate residue is found close to the thiol group as well, which is suggestive of deprotonation of the carbamoyl group at the beginning of the reactions.     

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K_M of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K_M observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.     

Defining the ATPase center of bacteriophage T4 DNA packaging machine: requirement for a catalytic glutamate residue in the large terminase protein gp17

Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID(251-255)) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.     

Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid

By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys(51)-SH to Cys(51)-SO(2)H. Thus, Cys(51)-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys(51)-SO(2)H, which cannot be reversed by thioredoxin. The presence of H(2)O(2) alone was not sufficient to cause oxidation of Cys(51) to Cys(51)-SO(2)H. Rather, the presence of complete catalytic components (H(2)O(2), thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys(172)/Ser(172) mutant Prx I required not only H(2)O(2), but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 microm) of H(2)O(2) indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 degrees C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H(2)O(2).     

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.     

ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.     

Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.     

Structural and functional regulation of eukaryotic 2-Cys peroxiredoxins including the plant ones in cellular defense-signaling mechanisms against oxidative stress

The ubiquitously distributed peroxiredoxins (Prxs) have been shown to have diverse functions in cellular defense‐signaling pathways. They have been largely classified into three Prx classes, 2‐Cys Prx, atypical 2‐Cys Prx and 1‐Cys Prx, which can be distinguished by how many Cys residues they possess and by their catalytic mechanisms. Proteins belonging to the typical 2‐Cys Prx group containing the N‐terminal peroxidatic Cys residue undergo a cycle of peroxide‐dependent oxidation to sulfenic acid and thiol‐dependent reduction during H₂O₂ catalysis. However, in the presence of high concentrations of H₂O₂ and catalytic components, including thioredoxin (Trx), Trx reductase and NADPH, the sulfenic acid can be hyperoxidized to cysteine sulfinic acid. The overoxidized 2‐Cys Prxs are slowly reduced by the action of the adenosine 5′‐triphosphate‐dependent enzyme, sulfiredoxin. Upon exposure of cells to strong oxidative or heat‐shock stress conditions, 2‐Cys Prxs change their protein structures from low‐molecular weight to high‐molecular weight complexes, which trigger their functional switching from peroxidases to molecular chaperones. The C‐terminal region of 2‐Cys Prx also plays an essential role in this structural conversion. Thus, proteins with truncated C‐termini are resistant to overoxidation and cannot regulate their structures or functions. These reactions are primarily guided by the active site peroxidatic Cys residue, which serves as an ‘H₂O₂‐sensor’ in cells. The reversible structural and functional switching of 2‐Cys Prxs provides cells with a means to adapt to external stresses by presumably activating intracellular defense‐signaling systems. In particular, plant 2‐Cys Prxs localized in chloroplasts have dynamic protein structures that undergo major conformational changes during catalysis, forming super‐complexes and reversibly attaching to thylakoid membranes in a redox‐dependent manner.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the γ-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.     

A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.