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pH-dependent activities and structural stability of loop-2-anchoring helix of RadA recombinase from Methanococcus voltae.

Rao DE, Luo Y - Protein Pept. Lett. (2014)

Bottom Line: Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH.We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA.His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. yu.luo@usask.ca.

ABSTRACT
RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein's ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA's pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

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ATPase Site in Stereo. Two recombinase subunits are coloured in yellow and grey, respectively. K+ ions, Mg2+ ions and water molecules are coloured in purple, red and green, respectively. The putative hydrolysis water in each structure is shown in a bigger sphere. Selected hydrogen bonds and metal-coordination bonds are shown in dashed lines in brown. A. ATPase site previously seen in a KCl-soaked crystal at pH 7.5. B. ATPase site seen in KCl-soaked crystals at pH 6.0. At the acidic pH, the direct contacts between the g-phosphate moiety of the ATP analogue and the electron-withdrawing potassium ions and His-280 side chain are lost.
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Figure 3: ATPase Site in Stereo. Two recombinase subunits are coloured in yellow and grey, respectively. K+ ions, Mg2+ ions and water molecules are coloured in purple, red and green, respectively. The putative hydrolysis water in each structure is shown in a bigger sphere. Selected hydrogen bonds and metal-coordination bonds are shown in dashed lines in brown. A. ATPase site previously seen in a KCl-soaked crystal at pH 7.5. B. ATPase site seen in KCl-soaked crystals at pH 6.0. At the acidic pH, the direct contacts between the g-phosphate moiety of the ATP analogue and the electron-withdrawing potassium ions and His-280 side chain are lost.

Mentions: Our previous soaking and co-crystallization experiments with MvRadA / AMP-PNP crystals in an ATPase-activating dose of a potassium salt at a pH range between 7.0 and 8.0 had reproducibly resulted in an ATPase-active conformation (PDB code 1XU4, Fig. 3A) with the highest quality data determined at 2.0 Å resolution [27, 46]. We were unable to get sizeable crystals at pH lower than 7.0, though showers of micro-crystals were observed. As a result, a soaking experiment using crystals grown at pH 7.5 was carried out. The ATPase activity of MvRadA in the presence of 0.4 M KCl is observed to be over 90% of the maximum reached at 0.8 – 1.6 M KCl. The crystals, however, could not survive soaking in stabilizing solutions with over 0.8 M KCl. Structures determined after soaking at pH 6.0 in the presence of 0.5 M KCl were also reproducible. Soaking in both Tris-Hepes (pH 6.0) and Mes-KOH (pH 6.0) buffers produced essentially identical structures except for differences in resolution from crystal to crystal. The resulting ATPase site is shown in (Fig. 3B). The best data set of pH 6.0 MvRadA / AMP-PNP complex soaked with Mes-KOH buffer was refined to 2.4 Å resolution. Compared with the ATPase-active filament at pH 7.5, the helical filament pitch of this pH 6.0 form is noticeably elongated from 105.4 to 107.2 Å. In both soaked crystals, the ATP analogue was tethered between MvRadA protomers. One subunit (yellow subunit, Fig. 3) binds the ATP analogue and an octahedral Mg2+ largely through its conserved P-loop (residues Gly-105 to Thr-112) [47] and the base-stacking Arg-158. The adjacent subunit (gray subunit, Fig. 3) contributes the ATP cap (residues Asp-302 to Asp-308) and the C-terminal portion of the L2 region (residues Asn-256 to Arg-285). In this manner, ATP bridges the filamentous assembly of MvRadA. The major differences lie in the L2 region, solvent entities near the triphosphate and positions of catalytic residues Glu-151 [13] and Gln-257 [48]. In the previously determined ATPase-active form at pH 7.5, two potassium ions and His-280 directly contact the γ-phosphate (2.6 – 2.9 Å), and both catalytic residues Glu-151 and Gln-257 form hydrogen bonds with a candidate for the nucleophilic water (big green sphere, Fig. 3A). We have interpreted these features as essential elements for promoting ATP hydrolysis. In the pH 6.0 structure, however, only one potassium ion (purple spheres, Fig. 3B) was located by anomalous signals between the γ-phosphate of the ATP analogue and the side chain of Asp-302. And the K+ ion was found too distant (3.4 Å) from the γ-phosphate to adequately polarize the phosphate for hydrolysis. The candidate for the nucleophilic water bound by Glu-151 was further away from the γ-phosphorous atom, with distance rising from 3.7 Å to 4.5 Å. It also lost hydrogen bond contact with the terminal phosphate. Unlike the largely ordered L2 region in the ATPase-active form, the entire L2 region in the pH 6.0 form was noticeably more disordered. There were no electron densities for a long stretch of residues from Ala-260 to Val-278. Notably, His-280 was located far apart from the ATP analogue. As such, the polarizing effect by His-280 is also lost. The catalytic side chains of Glu-151 and Gln-257 were found in different conformations. The Gln-257 side chain is too distant to form a hydrogen bond with the nucleophile candidate (big green sphere, Fig. 3B). The root-mean-square difference between the 300 ordered Cα atoms of the pH 6.0 and pH 7.5 structures is 0.65 Å. The deviation of the pH 6.0 structure from previously determined ATPase-inactive forms fall between 0.17 and 0.25 Å. Even for the ordered parts of the most flexible L2 region, the differences are within 1.0 Å. We conclude that the pH 6.0 conformation closely resemble the recurrent ATPase-inactive conformation determined in the presence of ADP or in the absence of a high concentration of a potassium salt [46]. Therefore, the pH 6.0 structure does not represent a novel conformation. Rather, we interpret the structural finding as suggesting the ATPase-active conformation is destabilized at pH 6.0.


pH-dependent activities and structural stability of loop-2-anchoring helix of RadA recombinase from Methanococcus voltae.

Rao DE, Luo Y - Protein Pept. Lett. (2014)

ATPase Site in Stereo. Two recombinase subunits are coloured in yellow and grey, respectively. K+ ions, Mg2+ ions and water molecules are coloured in purple, red and green, respectively. The putative hydrolysis water in each structure is shown in a bigger sphere. Selected hydrogen bonds and metal-coordination bonds are shown in dashed lines in brown. A. ATPase site previously seen in a KCl-soaked crystal at pH 7.5. B. ATPase site seen in KCl-soaked crystals at pH 6.0. At the acidic pH, the direct contacts between the g-phosphate moiety of the ATP analogue and the electron-withdrawing potassium ions and His-280 side chain are lost.
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Figure 3: ATPase Site in Stereo. Two recombinase subunits are coloured in yellow and grey, respectively. K+ ions, Mg2+ ions and water molecules are coloured in purple, red and green, respectively. The putative hydrolysis water in each structure is shown in a bigger sphere. Selected hydrogen bonds and metal-coordination bonds are shown in dashed lines in brown. A. ATPase site previously seen in a KCl-soaked crystal at pH 7.5. B. ATPase site seen in KCl-soaked crystals at pH 6.0. At the acidic pH, the direct contacts between the g-phosphate moiety of the ATP analogue and the electron-withdrawing potassium ions and His-280 side chain are lost.
Mentions: Our previous soaking and co-crystallization experiments with MvRadA / AMP-PNP crystals in an ATPase-activating dose of a potassium salt at a pH range between 7.0 and 8.0 had reproducibly resulted in an ATPase-active conformation (PDB code 1XU4, Fig. 3A) with the highest quality data determined at 2.0 Å resolution [27, 46]. We were unable to get sizeable crystals at pH lower than 7.0, though showers of micro-crystals were observed. As a result, a soaking experiment using crystals grown at pH 7.5 was carried out. The ATPase activity of MvRadA in the presence of 0.4 M KCl is observed to be over 90% of the maximum reached at 0.8 – 1.6 M KCl. The crystals, however, could not survive soaking in stabilizing solutions with over 0.8 M KCl. Structures determined after soaking at pH 6.0 in the presence of 0.5 M KCl were also reproducible. Soaking in both Tris-Hepes (pH 6.0) and Mes-KOH (pH 6.0) buffers produced essentially identical structures except for differences in resolution from crystal to crystal. The resulting ATPase site is shown in (Fig. 3B). The best data set of pH 6.0 MvRadA / AMP-PNP complex soaked with Mes-KOH buffer was refined to 2.4 Å resolution. Compared with the ATPase-active filament at pH 7.5, the helical filament pitch of this pH 6.0 form is noticeably elongated from 105.4 to 107.2 Å. In both soaked crystals, the ATP analogue was tethered between MvRadA protomers. One subunit (yellow subunit, Fig. 3) binds the ATP analogue and an octahedral Mg2+ largely through its conserved P-loop (residues Gly-105 to Thr-112) [47] and the base-stacking Arg-158. The adjacent subunit (gray subunit, Fig. 3) contributes the ATP cap (residues Asp-302 to Asp-308) and the C-terminal portion of the L2 region (residues Asn-256 to Arg-285). In this manner, ATP bridges the filamentous assembly of MvRadA. The major differences lie in the L2 region, solvent entities near the triphosphate and positions of catalytic residues Glu-151 [13] and Gln-257 [48]. In the previously determined ATPase-active form at pH 7.5, two potassium ions and His-280 directly contact the γ-phosphate (2.6 – 2.9 Å), and both catalytic residues Glu-151 and Gln-257 form hydrogen bonds with a candidate for the nucleophilic water (big green sphere, Fig. 3A). We have interpreted these features as essential elements for promoting ATP hydrolysis. In the pH 6.0 structure, however, only one potassium ion (purple spheres, Fig. 3B) was located by anomalous signals between the γ-phosphate of the ATP analogue and the side chain of Asp-302. And the K+ ion was found too distant (3.4 Å) from the γ-phosphate to adequately polarize the phosphate for hydrolysis. The candidate for the nucleophilic water bound by Glu-151 was further away from the γ-phosphorous atom, with distance rising from 3.7 Å to 4.5 Å. It also lost hydrogen bond contact with the terminal phosphate. Unlike the largely ordered L2 region in the ATPase-active form, the entire L2 region in the pH 6.0 form was noticeably more disordered. There were no electron densities for a long stretch of residues from Ala-260 to Val-278. Notably, His-280 was located far apart from the ATP analogue. As such, the polarizing effect by His-280 is also lost. The catalytic side chains of Glu-151 and Gln-257 were found in different conformations. The Gln-257 side chain is too distant to form a hydrogen bond with the nucleophile candidate (big green sphere, Fig. 3B). The root-mean-square difference between the 300 ordered Cα atoms of the pH 6.0 and pH 7.5 structures is 0.65 Å. The deviation of the pH 6.0 structure from previously determined ATPase-inactive forms fall between 0.17 and 0.25 Å. Even for the ordered parts of the most flexible L2 region, the differences are within 1.0 Å. We conclude that the pH 6.0 conformation closely resemble the recurrent ATPase-inactive conformation determined in the presence of ADP or in the absence of a high concentration of a potassium salt [46]. Therefore, the pH 6.0 structure does not represent a novel conformation. Rather, we interpret the structural finding as suggesting the ATPase-active conformation is destabilized at pH 6.0.

Bottom Line: Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH.We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA.His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. yu.luo@usask.ca.

ABSTRACT
RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein's ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA's pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

Show MeSH
Related in: MedlinePlus