The importance of the Q motif in the ATPase activity of a viral helicase

NS3 proteins of flaviviruses contain motifs which indicate that they possess protease and helicase activities. The helicases are members of the DExD/H box helicase superfamily and NS3 proteins from some flaviviruses have been shown to possess ATPase and helicase activities in vitro. The Q motif is a recently recognised cluster of nine amino acids common to most DExD/H box helicases which is proposed to regulate ATP binding and hydrolysis. In addition a conserved residue occurs 17 amino acids upstream of the Q motif ('+17'). We have analysed full-length and truncated NS3 proteins from Powassan virus (a tick-borne flavivirus) to investigate the role that the Q motif plays in the hydrolysis of ATP by a viral helicase. The Q motif appears to be essential for the activity of Powassan virus NS3 ATPase, however NS3 deletion mutants that contain the Q motif but lack the '+17' amino acid have ATPase activity albeit at a reduced level.     

Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action

The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The roles of two such motifs are examined here. The first motif (YRGXDV) forms a loop that connects SF2 helicase motifs 4 and 5, at the tip of which is Arg393. When Arg393 is changed to Ala, the resulting protein (R393A) retains a nucleic acid stimulated ATPase but cannot unwind RNA. R393A also unwinds DNA more slowly than wild type and translocates poorly on single-stranded DNA (ssDNA). DNA and RNA stimulate ATP hydrolysis catalyzed by R393A like the wild type, but the mutant protein binds ssDNA more weakly both in the presence and absence of the non-hydrolyzable ATP analog ADP(BeF3). The second motif (DFSLDPTF) forms a loop that connects two anti-parallel sheets between SF2 motifs 5 and 6. When Phe444 in this Phe-loop is changed to Ala, the resulting protein (F444A) is devoid of all activities. When Phe438 is changed to Ala, the protein (F438A) retains nucleic acid-stimulated ATPase, but does not unwind RNA. F438A unwinds DNA and translocates on ssDNA at about half the rate of the wild type. Equilibrium binding data reveal that this uncoupling of ATP hydrolysis and unwinding is due to the fact that the F438A mutant does not release ssDNA upon ATP binding like the wild type. A model is presented explaining the roles of the Arg-clamp and the Phe-loop in the unwinding reaction.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys693 and Asp714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn(2+), Pb(2+) and Cd(2+) out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K(693) and D(714), in transmembrane helices 7 and 8, were also addressed. The mutation CAAC-->SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P(i) is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC-->SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K(693) and D(714): the substitution K693N eliminates the Zn(2+)-stimulated ATPase activity completely, although significant Zn(2+)-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P(i). ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P(i). In conclusion, K(693) and D(714) are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

The Nucleoside Triphosphatase and Helicase Activities of Vaccinia Virus NPH-II Are Essential for Virus Replication

Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-ts viruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.     

Functional analysis of the trypanosomal AAA protein TbVCP with trans-dominant ATP hydrolysis mutants

TbVCP is a member of the AAA (ATPases Associated with a variety of cellular Activities) family of proteins containing two ATPase domains. Southern analysis indicates TbVCP to have a single-locus, two-copy, genomic organization. One copy, but not both, can be disrupted by targeted gene replacement, suggesting that TbVCP is essential for trypanosome viability. Site-directed mutagenesis of the ATP hydrolysis motifs indicates that the second conserved ATPase domain is essential for TbVCP activity. Constitutive overexpression of TbVCP with a single mutation in the second hydrolysis motif or with mutations in both hydrolysis motifs was not possible. Regulated overexpression of these mutants resulted in cell death as a dominant negative phenotype. In each case cell growth arrested at 24-h post-induction and at all stages of the cell cycle as judged by replication of nuclear and kinetoplast genomes. Onset of growth arrest coincided with the development of severe and characteristic morphological alterations for each mutant. Neither constitutive nor regulated overexpression of wild type TbVCP or the single first hydrolysis domain mutant had any overt effect on cell viability or morphology. However, the distinct phenotype of the double mutant indicates that the first hydrolysis domain, although not essential, does modulate overall TbVCP function. Finally, yeast complementation studies demonstrated that TbVCP can functionally replace the yeast homologue Cdc48p, indicating that protein.protein interactions essential to function have been maintained over great phylogenetic distances.     

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.     

Functional evaluation of bacteriophage T4 Rad50 signature motif residues

The repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome, and organisms have evolved a conserved mechanism to facilitate their repair. In eukaryotes, archaea, and some bacteriophage, a complex made up of Mre11 and Rad50 (MR complex), which are a nuclease and ATPase, respectively, is involved in the initial processing of DSBs. Rad50 is a member of the ATP Binding Cassette (ABC) protein superfamily, the members of which contain an important Signature motif that acts in trans to complete the dimeric ATP binding site. To explore the functional relevance of this motif, four of its five residues were mutated in bacteriophage T4 Rad50, and their respective ATPase and nuclease activities were evaluated. The mutations reveal the functional roles of the Signature motif in ATP binding, hydrolysis, and cooperativity. In several mutants, the degree of DNA activation of ATP hydrolysis activity is reduced, indicating that the Signature motif is involved in allosteric signal transmission between the DNA and ATP binding sites of the MR complex. ATP hydrolysis is not required for nuclease activity when the probe is near the beginning of the DNA substrate; however, when an internal probe is used, decreases in ATPase activity have substantial effects on nuclease activity, suggesting that ATP hydrolysis is involved in translocation of the complex. Unexpectedly, the ATP hydrolysis and nuclease activities are not directly correlated with each other, and each mutation appears to differentially affect the exonuclease activity of Mre11.     

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created 'super-repressors', in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA's repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.     

Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.     

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.     

Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.     

Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.     

Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function

Saccharomyces cerevisiae Prp43 is a DEAH-box RNA-dependent ATPase that catalyzes the release of excised lariat intron from the mRNA spliceosome. Previous studies identified mutations in Prp43 motifs I, II, and VI that were lethal in vivo and ablated ATP hydrolysis in vitro. Such Prp43 mutants exerted dominant-negative growth phenotypes when expressed in wild type cells and blocked intron release in vitro when added to yeast splicing extracts. Here, we assessed the effects of alanine and conservative substitutions at conserved residues in motifs Ia ((146)TQPRRVAA(153)), IV ((307)LLFLTG(312)), and V ((376)TNIAETSLT(384)) and thereby identified Arg150 (motif Ia), Phe309 (motif IV), Thr376, Leu383, and Thr384 (motif V) as being important for Prp43 function in vivo. Motif V mutations T376V, T384A, and T384V were lethal and dominant negative in vivo, and the mutant proteins inhibited lariat release in vitro. The T384A and T384V proteins were proficient for ATP hydrolysis, suggesting that ATPase activity is necessary, but not sufficient, for Prp43 function. We report that Prp43 hydrolyzes all common NTPs and dNTPs and unwinds short 5'/3' tailed RNA/DNA duplexes in an ATP-dependent fashion. Optimal ATP hydrolysis requires an RNA cofactor of >or=20 nt. Prp43 is largely indifferent to mutations in its C-terminal segment, which is conserved in the DEAH-box splicing factors Prp2, Prp16, and Prp22.     

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.     

Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli

DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria. It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules. The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism. We constructed MBP-tagged GyrB mutants I10G and Delta4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP. Binding of ADPNP to MBP-tagged mutants was significantly decreased. ADPNP had no effect on DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP-tagged wild type enzyme. Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficiency and DNA transfer through DNA gyrase.     

ATP binding to hemoglobin response gene 1 protein is necessary for regulation of the mating type locus in Candida albicans

HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTLα gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) ～2 μM. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its γ-phosphate. ATP bound somewhat more avidly than ATPγS to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTlα2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.     

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.     

Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication.

The six MCM (minichromosome maintenance) proteins are essential DNA replication factors that each contain a putative ATP binding motif and together form a heterohexameric complex. We show that these motifs are required for viability in vivo and coordinated ATP hydrolysis in vitro. Mutational analysis discriminates between two functionally distinct MCM protein subgroups: Mcm4p, 6p, and 7p contribute canonical ATP binding motifs essential for catalysis, whereas the related motifs in Mcm2p, 3p, and 5p serve a regulatory function. Reconstitution experiments indicate that specific functional interactions between these two subgroups are required for robust ATP hydrolysis. Our observations show parallels between the MCM complex and the F1-ATPase, and we discuss how ATP hydrolysis by the MCM complex might be coupled to DNA strand separation.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys and Asp in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn²⁺, Pb²⁺ and Cd²⁺ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K⁶⁹³ and D⁷¹⁴, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P_i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K⁶⁹³ and D⁷¹⁴: the substitution K693N eliminates the Zn²⁺-stimulated ATPase activity completely, although significant Zn²⁺-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P_i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P_i. In conclusion, K⁶⁹³ and D⁷¹⁴ are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.