The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.     

Analysis on origin recognition complex containing Orc5p with defective Walker A motif

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.     

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotide's γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.     

The ATP Sites of AAA+ Clamp Loaders Work Together as a Switch to Assemble Clamps on DNA

Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.     

VAT, the thermoplasma homolog of mammalian p97/VCP, is an N domain-regulated protein unfoldase

The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.     

Structure and mechanism of the RuvB Holliday junction branch migration motor

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.     

Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast

The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.     

Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions. Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb.

Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69). K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro. In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.     

Characterization of a trap mutant of the AAA+ chaperone ClpB

The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE). The order of action of ClpB and KJE on aggregated proteins is unknown. We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP. This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time. Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB. The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions. ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates.     

Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly

Combinatorial association of DNA-binding proteins on composite binding sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein–protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 within the N-terminal β-hairpin motif of its paired domain. The β-hairpin also participates in recognition of a subset of Pax-5-binding sites. Thus, Pax-5 incorporates protein–protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Together, our results define specific amino acid requirements for Pax–Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein

The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg(359) and Arg(362) in the first AAA domain, D1 and Arg(635) and Arg(638) in the second AAA domain, D2. We show that Arg(359) and Arg(362) in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg(635) and Arg(638) can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg(359) and Arg(362) uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.     

Crystal structure of the SF3 helicase from adeno-associated virus type 2

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.     

Genetic and cell biological characterization of the vaccinia virus A30 and G7 phosphoproteins

The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents, and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo and recapitulate that modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during nonpermissive tsH5 infections, suggesting that the loss of A30/G7 is the underlying cause for the formation of lacy or curdled virosomes. We also determined that the temperature-sensitive phenotype of the Cts11 virus is due to mutations in two codons of the G7L gene. Phenotypic analysis of nonpermissive Cts11 infections indicated that these amino acid substitutions compromise G7 function without impairing the stability of either G7 or A30. Utilizing Cts11 in conjunction with a rifampin release assay, we determined that G7 acts at multiple stages of virion morphogenesis that can be distinguished both by ultrastructural analysis and by monitoring the phosphorylation status of several viral proteins that undergo F10-mediated phosphorylation.     

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.