Alanine scanning of UvrD1
The primary structure alignment in highlights 330 positions of side chain identity/similarity in M. smegmatis
UvrD1, E. coli
UvrD, and B. stearothermophilus
PcrA. UvrD1 has apparent counterparts of all of the ATPase, DNA binding, and domain interaction motifs enumerated for E. coli
). In our initial characterization of UvrD1 (7
), we found that ATP hydrolysis was abolished by a single-alanine substitution for the metal-binding motif II aspartate (Asp235) or a double-alanine replacement of the motif I residues Lys45 (which contacts the β and γ phosphates of ATP) and Thr46 (which coordinates the metal) (). Suppression of ATPase activity by the D235A change coordinately abolished duplex DNA unwinding, but did not affect the binding of UvrD1 to the 3’ tailed DNA helicase substrate (7
) (). Similar ablative effects of motif I and II mutations on catalysis and motor function, but not nucleic acid binding, have been reported for the PcrA DNA helicase (11
) and the SF2 RNA helicase NPH-II (14
), among others, which testifies to the universal importance of these two motifs found in all of the helicase superfamilies (1
). The goal of the present study was to extend the mutagenesis of UvrD1 to other residues that contact Mg2+
·ATP. In this effort, we were guided by the 2.2 Å structure of E. coli
) bound to a 3’ tailed duplex DNA, with the active site occupied by Mg2+
, comprising a mimetic of the pentacoordinate transition state of the phosphohydrolase reaction (). We targeted seven candidate active site residues for alanine scanning, including the UvrD1 counterparts of: (i) those that contact the ATP adenine base (Gln24 in the “Q motif”), the ribose sugar (Glu609 in motif V), or the a phosphate (Arg83 in motif Ia); and (ii) those that coordinate the nonbridging γ phosphate oxygens, the attacking water nucleophile, or the bridging β-γ oxygen leaving group (Glu236 in motif II, Gln275 in motif III, Arg308 in motif IV, Arg648 in motif VI) (). We also targeted the UvrD1 equivalents – Phe72 in motif Ia, Phe280 in motif III, and Trp664 in motif VIa – of three aromatic amino acids that directly contact the nucleosides of the 3’ single-stranded tail of the “loading strand” of the helicase substrate (). This is the nucleic acid segment to which the UvrD/PcrA-type helicases initially bind and along which they translocate in response to ATP hydrolysis (1
). Wild-type UvrD1 and the ten UvrD1-Ala mutants were produced in E. coli
and purified from soluble bacterial extracts by serial affinity and ion-exchange column chromatography steps to achieve comparable enrichments for each of the enzyme preparations ().
ATPase active site and DNA contacts
Effect of active site mutations on ATPase and helicase activity
The specific activities of the wild-type and mutant UvrD1 protein in DNA-dependent ATP hydrolysis were determined by protein titration under conditions optimized previously for wild-type UvrD1 (). The most severe mutational effects were elicited by alanine substitutions at Arg308, Arg648 and Gln275 – the very residues implicated in phosphohydrolase chemistry at the ATP γ phosphate (). The specific activities of the R308A and R648A proteins were ≤0.05% of the wild-type value (this being the limit of sensitivity of our assays), whereas Q275A was 0.3% as active as wild-type UvrD1 (). (These proteins were scored as “ATPase –” in , which summarizes the mutational results.) Alanine in lieu of Glu236 (which contacts the nucleophilic water and a water in the octahedral magnesium coordination complex) reduced specific activity to 7% of wild-type (scored as “one-plus” activity in , where wild-type activity is “three-plus”). By contrast, alanine substitutions at several residues that interact with the adenosine nucleoside had more benign effects on ATPase activity, ranging in magnitude from minimal (E609A, +++) to modest (R83A, Q24A, scored as ++ in ) ().
The helicase activity of the UvrD1 proteins was assayed in the presence of Ku and ATP using a 3’-tailed duplex substrate consisting of a 24-bp duplex with a 3’ T20
tail on the loading strand and a 5’ 32
P-label on the displaced strand (). The assay format entails preincubation of 110 nM UvrD1, 100 nM Ku, and 50 nM labeled DNA, followed by initiation of unwinding by addition of 1 mM ATP, with simultaneous addition of a “trap” of excess unlabeled displaced strand that: (i) minimizes reannealing of any labeled 24-mer that was unwound by UvrD1 and (ii) competes with the loading strand for binding to any free UvrD1 or UvrD1 that dissociated from the labeled DNA without unwinding it. Consequently, the assay predominantly gauges a single round of strand displacement by UvrD1 bound to the labeled duplex prior to the onset of ATP hydrolysis. As noted previously (7
), the combination of Ku and wild-type UvrD1 unwound the DNA to yield a radiolabeled free single strand that migrated faster than the input tailed duplex during native PAGE; the helicase reaction product comigrated with free 24-mer generated by thermal denaturation of the substrate (). As expected, helicase activity was suppressed by the four alanine mutations that ablated or severely reduced ATP hydrolysis: R308A, R648A, Q275A, and E236A, (; scored as “helicase –” in ). The R83A mutant, which retained substantial ATPase activity, also retained near wild-type helicase activity, whereas Q24A, which was less active in ATP hydrolysis, displayed concordantly reduced strand displacement activity (). The remarkable finding was that the E609A mutant was as defective in duplex unwinding as any of the “deadest” ATPase-defective mutants (), notwithstanding that E609A itself retained near wild-type DNA-dependent ATPase activity. This result could signify that Glu609 is required to couple ATP hydrolysis to the work-performing motor activity of UvrD1.
Effect of active site mutations on DNA binding
The ability of the UvrD1-Ala mutants to bind to the helicase substrate was gauged by native gel electrophoresis (). As noted previously, wild-type UvrD1 efficiently formed a discrete protein-DNA complex of retarded electrophoretic mobility. UvrD1-DNA complexes were formed in good yield by the R83A and Q24A mutants that retained helicase activity (scored as +++ DNA binding in ). The instructive findings concerned DNA binding by the other active site Ala-mutants, as follows. First, the formation of a discrete UvrD1-DNA complex was unperturbed by the R308A and R648A changes that abolished ATPase and helicase activity, implying that these two arginines are direct catalysts of ATP hydrolysis by DNA-bound UvrD1. Second, the Q275A and E236A changes diminished UvrD1 binding to the helicase substrate (). The reduction in DNA binding by Q275A was partial (scored as ++ DNA binding in ), and by no means commensurate with its ablation of ATP hydrolysis, which we presume is the dominant factor in the loss of helicase activity. It is conceivable that Q275A exerts an effect on DNA binding via local changes in motif III, especially affecting neighboring DNA-binding residues such as Tyr278 and Phe280 (). The more pronounced negative effect of the E236A mutation on UvrD1 binding to the helicase substrate (scored as “↓↓” binding in ) precludes simple attribution of the E236A unwinding defect to a decrement in ATP hydrolysis. (Note that the loss of the vicinal Asp235 motif II side chain had no apparent effect on DNA binding in this assay (7
); .) The UvrD/PcrA structures suggest a possible connection between the conserved motif II glutamate and DNA binding, whereby the glutamate forms a salt bridge from Oε1 (the carboxylate oxygen not engaged to Mg2+
·ATP) to a conserved motif V lysine (Lys563 in UvrD; Lys606 in UvrD1, Lys568 in PcrA). This contact appears to help tether a short protein α-helix near the DNA 3’ tail, from which a conserved motif V histidine (His560 in UvrD, His603 in UvrD1, His565 in PcrA) makes direct contact with the DNA and also donates a hydrogen bond to a main chain carbonyl in the DNA-binding segment of motif III (3
The third and most informative finding from gel-shift assays of DNA binding by the active site mutants was that the E609A change had no apparent impact on stable UvrD1 binding to the helicase substrate (). This signifies that Glu609 does indeed couple ATP hydrolysis to mechanical work.
As another means of assessing effects on DNA binding, we measured the extent of ATP hydrolysis as a function of increasing concentration of the input DNA cofactor. (This assay was only applicable to UvrD1 active site mutants that retained appreciable ATPase activity under standard assay conditions using 5 µg/ml salmon sperm DNA.) Wild-type UvrD1 ATPase displayed a hyperbolic dependence on DNA with an apparent Km of 0.16 µg/ml and Vmax of 170 s−1. The values for Ala-mutants were as follows: E609A (0.24 µg/ml, 170 s−1); R83A (0.34 µg/ml, 62 s−1); Q24A (0.55 µg/ml, 45 s−1); E236A (1.7 µg/ml, 8 s−1). These results fortify the inferences from the ATPase enzyme titration experiments and native gel DNA binding assays that: (i) Glu609 is not important for ATP hydrolysis or DNA binding, (ii) Arg83 and Gln24 enhance ATP hydrolysis 3- to 4-fold, and impact modestly on DNA binding, and (iii) Glu236 assists both ATP hydrolysis and DNA binding, e.g., the Km of E236A for sperm DNA was 10-fold higher than that of wild-type UvrD1.
Effects of conservative mutations in the ATPase active site
Structure-activity relationships for selected active site residues were gleaned by studying the effects of conservative amino acid substitutions. Arg83, Arg308 and Arg648 were replaced by lysine and glutamine; Glu236 was changed to glutamine; Glu609 was replaced by glutamine and aspartate; Gln275 was mutated to asparagine, lysine, and arginine. The conservative active site mutants were purified () and surveyed for DNA-dependent ATPase () and DNA unwinding () activities. We found that Arg648 was strictly essential, insofar as neither lysine nor glutamine restored ATPase or helicase activity vis a vis the catalytically defective R648A mutant (). Similar findings pertained to Arg308 (). We surmise that positive charge is not sufficient and that ATP hydrolysis requires the multidentate contacts of Arg308 and Arg648 with the ATP phosphate oxygens, especially in stabilizing the extra negative charge developed on the γ phosphate in the predicted associative transition state (). Different structure-activity relations were seen at Arg83, whereby the modest defect in ATP hydrolysis by the R83A mutant was mimicked by the glutamine substitution, while activity was improved to wild-type level when lysine was introduced (). We surmise that the positive charge on motif Ia residue Arg83 is the relevant factor in its helpful contact with the ATP α phosphate (). As expected, the R83K and R83Q proteins retained DNA unwinding activity ().
Effects of conservative mutations on UvrD1 activities
Replacing Glu236 with glutamine effectively abolished ATPase activity (E236Q specific activity was ≤0.05% of the wild-type value) and was more deleterious in this regard than the E236A change. Mutation of Gln275 to asparagine elicited as severe a decrement in ATP hydrolysis as the alanine substitution, signifying that a critical distance from the main-chain to the amide functional group is required to achieve the contacts with the water nucleophile and the γ phosphate seen in the UvrD crystal structure (). Lysine and arginine were equally damaging in lieu of Gln275 (). These results underscored the strict reliance for ATP hydrolysis on the conserved motif III Gln and motif II Glu residues that together orient (and activate) the attacking water. As expected, the ATPase-defective Gln275 mutants were also defective for DNA unwinding ().
Finally, we found that replacing Glu609 with either aspartate or glutamine preserved ATPase activity (), as noted for the E609A mutant. Whereas E609D had no salutary effect on DNA unwinding, E609Q partially restored helicase activity (). We infer that coupling of UvrD1 ATP hydrolysis to duplex unwinding depends on a critical distance from the main chain to the Glu609 carboxylate that allows the hydrogen bonds to the ATP ribose O3’ seen in the UvrD crystal structure (). Glutamine, a bivalent hydrogen bond donor/acceptor, could in principle engage in the same ribose contacts.
The Q motif serves as an adenine nucleotide specificity filter in UvrD1
The Gln24 residue of UvrD1 comprises the “Q motif” located 21- to 24-aa upstream of the motif I lysine in a wide variety of nucleic acid-dependent NTP phosphohydrolases (15
), including SF1 helicases UvrD, PcrA and Rep (2
), the DExH-box protein UvrB (17
), the DEAD-box RNA helicases eIF4A, Ded1, Dhh1, and Hera (15
), and many others. In the adenine nucleotide-bound crystal structures of UvrD (4
), UvrB (17
) and Hera (20
), the Q motif glutamine makes bidentate hydrogen bonds from Gln-Nε to the adenine N7 atom and from Gln-Oε to the adenine N6 atom (). The Q motif glutamine has been touted as having a “regulatory” function in ATP hydrolysis and nucleic acid binding (15
), but there is as yet no coherent view of what is being regulated and how, given that alanine mutations of the Q motif glutamine exert very strong suppressive effects on the phosphohydrolase activities of several different ATPase enzymes, including eIF4A (15
), Ded1 (16
) and reverse gyrase (21
). Such findings suggest a simple requirement for the Q motif glutamine for avid binding of ATP (and hence vigorous hydrolysis), in keeping with the crystallographic contacts. It has been speculated that the Q motif glutamine might enforce specificity for adenine nucleotides (15
), but there is scant evidence in support of this idea.
In light of our initial findings that the UvrD1 Q24A mutant retained substantial DNA-dependent ATPase activity (one fourth that of wild-type), we viewed UvrD1 as a promising model to test the regulatory potential of the Q motif. Mycobacterial UvrD1 specifically utilizes adenosine nucleotides for the phosphohydrolase and helicase reactions (7
). Wild-type UvrD1 readily hydrolyzed either ATP or dATP in the presence of salmon sperm DNA; other rNTPs and dNTPs were hydrolyzed poorly (). By contrast, the Q24A mutant was able to hydrolyze all rNTP and dNTPs (). Thus, the loss of the glutamine contacts to the adenine base elicited a gain-of-function, e.g., whereby UTP and TTP (the worst substrates for wild-type UvrD1) were now on a par with ATP and dATP as substrates for Q24A.
Role of the Q motif (Gln24) in NTP substrate specifity
These inferences from single-point assays were fortified by nucleotide titration experiments. The steady-state kinetic parameters of wild-type UvrD1 for ATP hydrolysis were: Km 0.26 ± 0.04 mM ATP, kcat 265 ± 22 s−1. The values for the Q24A mutant were: Km 0.69 ± 0.05 mM ATP, kcat 95 ± 3 s−1. Thus, the Q motif glutamine enhances affinity of UvrD1 for ATP by a mere 3-fold, with a similar contribution to the rate of catalysis. The steady state parameters for UTP hydrolysis by Q24A were: Km 1.21 ± 0.13 mM UTP, kcat 86 ± 5 s−1. There was scant difference in the binding or hydrolysis of ATP versus UTP by the Q24A enzyme. These results provide evidence of a regulatory role for the Q motif as a substrate specificity filter.
Assays of helicase activity suggested an additional level of control by the Q motif. Wild-type UvrD1 unwound the tailed duplex in the presence of ATP, but not UTP (). Q24A again had lower than wild-type strand displacement activity in the presence of ATP or dATP (). The instructive findings were that Q24A retained its specificity for adenosine nucleotides in the helicase reaction, notwithstanding its relaxed nucleotide specificity in the phosphohydrolase reaction. In particular, Q24A, like wild-type UvrD1, was ineffective as a helicase in the presence of UTP (). We infer that the Q motif glutamine plays a role in coupling NTP hydrolysis to mechanical work.
Effects of mutations of putative DNA-binding residues Phe72, Phe280 and Trp664
Alanine mutations were introduced at the UvrD1 counterparts of aromatic amino acids in UvrD/PcrA that contact the nucleosides of the 3’ tail of the loading strand (). The recombinant F72A, F280A, and W664A proteins () exerted disparate effects on ATP hydrolysis, ranging from comparatively severe (F280A) to modest (F72A) to none (W664A) (, ). We measured the extent of ATP hydrolysis by W664A as a function of increasing concentrations of DNA, as described above for the active site mutants, and observed an apparent Km of 0.47 µg/ml salmon sperm DNA and a Vmax of 180 s−1 (values quite similar to wild-type UvrD1 in the same assay format). W664A was able to unwind the 3’ tailed duplex () and formed a stable complex with the helicase substrate (). We surmise that the putative contacts of Trp664 with the loading strand near the single-strand/duplex junction are not crucial for any of the UvrD1 activities tested. Alternatively, it is possible that UvrD1 Trp664 does not contact the DNA in the manner surmised from the E. coli UvrD crystal structure.
The F280A mutant hydrolyzed ATP with an apparent Km
of 2.3 µg/ml salmon sperm DNA (15-fold higher than wild-type UvrD1) and a Vmax
of 4 s−1
. F280A was defective in duplex unwinding () and failed to form a stable complex with the helicase substrate in the native gel-shift assay (). The aromatic motif III residue equivalent to Phe280 in UvrD1/PcrA (; ) forms a π stack on one of the bases of the loading strand (). A key tenet of the translocation mechanism proposed for SF1 helicases (1
) entails ATP-driven flipping of the single-stranded bases of the loading strand between sandwiching aromatic residues, especially those of motif III. Replacing UvrD1 Phe280 with leucine resulted in a partial gain of function in ATP hydrolysis compared to the Ala-mutant (to about one-fourth of wild-type specific activity; ) that was accompanied by a slight recovery of DNA unwinding activity ().
F72A hydrolyzed ATP with an apparent Km of 0.23 µg/ml salmon sperm DNA and a Vmax of 33 s−1. F72A formed a stable complex with the helicase substrate (), yet was defective in duplex unwinding (). The F72A helicase defect was apparently more pronounced than the decrement in ATPase activity (e.g., F72A and Q24A had similar activities in ATP hydrolysis and DNA binding, yet only Q24A had partial unwinding activity), which suggests that Phe72 aids in coupling ATP consumption to motor function. Replacing Phe72 with leucine afforded no improvement in ATPase activity compared to F72A (), but did allow a trace amount of DNA unwinding ().
Effects of C-terminal deletions of UvrD1
The C-terminal domains (~70-aa) of UvrD and PcrA are either disordered or missing in the crystal structures of the respective DNA-bound enzymes (3
). In the case of E. coli
UvrD, deletion of 102-aa from the C-terminus ablated all its biochemical activities, whereas a 40-aa C-terminal truncation did not affect the ATPase and helicase functions (27
). Here we studied the effects of three incremental C-terminal deletions on the activities of mycobacterial UvrD1. The full-length UvrD1-(1–783) protein was compared to C-terminal truncation mutants UvrD1-(1–729), UvrD1-(1–693) and UvrD1-(1–595). SDS-PAGE verified the anticipated decrements in the apparent sizes of the truncated polypeptides (). UvrD1-(1–729) and UvrD1-(1–693) were as active as full-length UvrD1 in DNA-dependent ATP hydrolysis (). By contrast, UvrD1-(1–595) was virtually catalytically inert (); this was expected, given that this truncation mutant lacks the essential active site Arg648 residue of motif VI (). Although, the UvrD1-(1–729) and UvrD1-(1–693) proteins retained strand displacement activity in the presence of Ku, the extents of unwinding in the single-turnover helicase assay decreased progressively with each deletion increment (, reflected in the higher levels of residual unwound duplex). The full-length UvrD1-(1–783) and truncated UvrD1-(1–693) proteins were equally adept at binding stably to the 3’-tailed helicase substrate in the native gel-shift assay (). The binary DNA-UvrD1-(1–693) complex migrated more rapidly than that of the full-length enzyme, consistent with the reduced mass of the truncated UvrD1 protein. The instructive finding was that whereas full-length UvrD1-(1–783) formed a stable supershifted DNA-UvrD1-Ku ternary complex when Ku was included in the binding reaction, UvrD1-(1–693) did not (). UvrD1-(1–729) behaved similarly to UvrD1-(1–693) in the DNA binding assay (not shown). We surmise that that the C-terminus of UvrD1 enhances its stable interaction with Ku, although it is not strictly required for functional Ku-UvrD1 interactions, insofar as UvrD1-(1–693) helicase activity is still responsive to Ku stimulation.
Effects of C-terminal deletions
Influence of 3’ tail length on DNA unwinding by UvrD1
In the UvrD/PcrA structures, the helicase contacts a five-nucleotide segment of the 3’ single-stranded tail of the loading DNA strand and a 14–16 bp duplex DNA segment (2
). To probe the 3’-tail requirement for duplex unwinding by mycobacterial UvrD1, we tested a series of helicase substrates with 3’-oligo(dT) tails of varying length (20, 15, 10, or 5 nucleotides) attached to an identical 24-bp duplex segment. We found that UvrD1-mediated displacement of the radiolabeled 24-mer strand in the presence of Ku was optimal when the 3’ tail length was 20 nucleotides (). Helicase activity decreased slightly when the 3’ tail was retracted to 15 nucleotides, and then declined acutely when the tail was shortened to only 10 or 5 nucleotides. Analysis of UvrD1 binding to the helicase substrates by native gel electrophoresis revealed efficient formation of stable DNA-UvrD1 binary complexes whether the tail was 20, 15, 10 or 5 nucleotides (). As noted above, inclusion of Ku in the binding reaction mixture resulted in the formation of a stable supershifted DNA-Ku-UvrD1 ternary complex (). By contrast, the binding of Ku alone to the tailed duplex was metastable, yielding scant amounts of a slowly migrating Ku-DNA binary complex (indicated at right in ), plus a diffuse smear of radiolabeled material migrating just above the free DNA. As suggested previously (7
), the Ku-DNA binary complex is prone to dissociate during electrophoresis as the ringshaped Ku dimer (28
) slides off the short duplex segment of the DNA ligand. The pertinent findings here were that UvrD1 formed a discrete supershifted ternary complex with Ku when the 3’ single-stranded tail was shortened to 15 nucleotides, but not when the tail was further recessed to 10 or 5 nucleotides (). In the latter two cases, we see that Ku converted the discrete DNA-UvrD1 binary complexes to a diffuse species migrating just slower than the binary complex. We surmise that the ternary complex is less stable when the 3’ tail is 10 or 5 nucleotides. Therefore, the decrement in duplex unwinding upon shortening the 3’ tail is explained by an attenuation of Ku stimulation, not a primary effect on UvrD1 binding to the helicase substrate. We speculate that UvrD1 might bind closer to the duplex segment of the DNA when the 3’ single-strand tail is shortened, thus providing less room for Ku to embrace the duplex segment.
Effect of 3’ tail length on helicase activity and DNA binding