Abstract
Catalysis by members of the RNase H superfamily of enzymes is generally believed to require only two Mg2+ ions that are coordinated by active-site carboxylates. By examining the catalytic process of Bacillus halodurans RNase H1 in crystallo, however, we found that the two canonical Mg2+ ions and an additional K+ failed to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation required a second K+ and a third Mg2+, which replaced the first K+ and departed immediately after cleavage. A third transient Mg2+ has also been observed for DNA synthesis, but in that case it coordinates the leaving group instead of the nucleophile as in the case of the RNase H1 hydrolysis reaction. These transient cations have no contact with the enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand-transfer reactions in a single active site may similarly require cation trafficking coordinated by the substrate.
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Acknowledgements
We are grateful for the help provided by D. Kaufman and L. Wise in RNase H1 protein preparation and co-crystallization. We thank L. Tabak for generous support to N.L.S., and R. Craigie, F. Dyda, M. Gellert and D. Leahy for critical reading of the manuscript. This research was supported by the NIH Intramural AIDS-targeted Antiviral Program (IATAP), the NIDDK (DK036144-11; W.Y. and N.L.S.) and the NIDCR (N.L.S. via L. Tabak).
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N.L.S. and W.Y. designed the experiments; N.L.S. performed the experiments; and N.L.S. and W.Y. interpreted the results and wrote the manuscript.
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Supplementary Figure 1 RNA hydrolysis in crystallo.
a, A diagram of co-crystallization of RNase H1 and RNA:DNA hybrid, procedure of in crystallo reaction and preparation for X-ray diffraction data acquisition. b,c, Structures of EGTA-soaked RNase H1–substrate co-crystals with data collected at λ = 1.0 Å and λ = 1.54 Å. Altered conformations of D71 and E109 in the absence of two canonical Me2+ ions are shown with the pink omit map (contoured at 3σ), and the reference structure in the presence of Mg2+ ions is shown as semitransparent gray sticks (D71) and spheres (Mg2+). c, The presence of K+ after EGTA soaking is confirmed by the anomalous signal (contoured at 3σ) at λ = 1.54 Å.
Supplementary Figure 2 Monovalent cations in RNase H1 catalysis.
a, Solution analysis showing that Na+, K+ and Rb+ support the RNase H1 reaction, but Li+ causes much reduced catalysis. Here n = 3 independent experiments. The plotted values are the mean, and error bars represent 1 s.d. b, Identification of U, V and W monovalent cations. K+ was substituted by Rb+ in the in crystallo reaction, and the reaction process is shown at t = 40 s and 120 s after exposure to 5 mM Mg2+. The substrate (yellow) and product (blue) structures are superimposed with the pink Fo – Fc map (omitting the scissile-phosphate, contoured at 5.5σ) and Rb+ anomalous map (golden mesh, contoured at 4.5σ for the U and V sites and 8σ for the W site). c, During the reaction time course, U-site occupancy decreased with increase in product formation. d, Li+ persisted in occupying the A and B sites after a 120-s soak in 2 mM Mg2+. This structure is superimposable on the structure before soaking in Mg2+. Even though no 2Fo – Fc density (gray mesh) for Li+ could be observed at 1 ▯, the omit Fo – Fc map (magenta mesh) clearly shows the presence of Li+ in the A and B sites. e, Comparison of the W-site K+/Rb+ in RNase H1 and the C-site Mn2+ in DNA Pol η in an orthogonal view of Fig. 2c.
Supplementary Figure 3 Evidence for a third Me2+ in wild-type RNase H1 catalysis.
a, The in crystallo reaction catalyzed by WT RNase H1 was 80% complete with 20 mM Mg2+ at t = 40 s. E188 and K196 interact with each other. E188 also interacts with the A-site Mg2+ ion via a water molecule, and K196 interacts with the 5´-phosphate product. b, Dependence of RNase H1 catalytic rates on Mn2+ concentrations in solution. Initially increased concentration of Me2+ increases the reaction rate, probably owing to the low affinity of C-site Me2+, but high concentrations of Me2+ eventually lead to prolonged binding of Me2+C and prevent product release. Here n = 3 independent experiments. The plotted values are the mean, and error bars represent 1 s.d. c, In the in crystallo reaction, the A and B occupancies (magenta) and product formation (blue) required different Mn2+ concentrations. d, The in crystallo reaction catalyzed by WT RNase H1 was 100% complete with 500 mM Mn2+ at t = 40 s. K196 is disordered in this state, and Mn2+C (magenta) occupies that space and similarly interacts with the 5´-phosphate product, as well as the downstream phosphate and four water ligands. e, Mn2+C (magenta) also appears in the in crystallo reaction catalyzed by RNaseH1E188A at t = 240 s, where K196 is similarly disordered despite the presence of product. f, One of the water ligands of Mn2+C overlaps with K+U, and another water ligand of Mn2+C overlaps with K+V, both enclosed in an oval. If present, K196 (shown as semitransparent sticks) overlaps with Mn2+C.
Supplementary Figure 4 Defects of K196A mutant RNase H1.
a, In crystallo reactions with 4 mM Mn2+ rescued the slow reaction and most product inversion with the K196A mutation. The C-site Mn2+ is superimposable with that in the reaction catalyzed by RNaseH1E188A (Fig. 4b). The golden anomalous map (contoured at 3.5σ) and the pink Fo – Fc omit map (contoured at 6σ) are also shown. b, In the in crystallo catalysis by RNaseH1K196A, the 5´-phosphate product (blue) was inverted and adopted the same conformation as the substrate (yellow) by t = 720 s and most clearly at t = 1,800 s. The pink omit Fo – Fc map (5σ) is superimposed onto the structural model. The three monovalent cations were confirmed by Rb+ anomalous signals (golden mesh, contoured at 12σ for Rb+W and 3.5σ for Rb+U and Rb+v) as with WT RNase H1. c, Presence of a positively charged residue (K196 equivalent) adjacent to the C-terminal catalytic carboxylate is conserved in RNase H1 homologs.
Supplementary Figure 5 Defects of E188A mutant RNase H1.
a, In crystallo catalysis by E188A RNase H1 reveals delayed binding of the A-site Mg2+, which took 200 s to displace K+ ions instead of 40 s as in WT RNase H1 (shown in gray). b, By t = 360 s, the C-site Mg2+ along with its coordination ligands and the shifted 5´-phosphate product were observed as WT enzyme in the presence of Li+/Mg2+. Structures in a and b are superimposed with pink Fo – Fc omit maps contoured at 6σ for 200 s and 3.5σ for 360 s. c, Comparison of the third Mg2+ in the reactions catalyzed by WT (Li+) (shown in gray) and E188A mutant RNase H1. For the WT structure, Li+ was modeled in the B site because it had much lower 2Fo – Fc signal than the A site while the five coordination ligands were the same as when the B site was fully occupied.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5
Supplementary Video 1
Animation of the RNA cleavage reaction catalyzed by RNase H1. The crystal structures of WT RNase H–RNA:DNA complexes soaked in 2 mM Mg2+ for 40 s, in which two Mg2+ ions and K+U are bound (the misaligned substrate state), and for 480 s, in which the aligned substrate and product state coexist, are morphed using LSQMAN. The Mg2+C position coordinated by the 5′-phosphate product is borrowed from Mn2+C in the structure of the E188A crystal soaked in 4 mM Mg2+ for 240 s. The initial positions of Mg2+C, K+V and K+W as well as the end positions of Mg2+C, K+U and K+V were randomly chosen. The nucleophilic water is shown as a red sphere, and other Mg2+-coordinating water molecules are shown as pink spheres.
Supplementary Dataset 1
Source data for Fig. 4a
Supplementary Dataset 2
Data collection and refinement statistics
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Samara, N.L., Yang, W. Cation trafficking propels RNA hydrolysis. Nat Struct Mol Biol 25, 715–721 (2018). https://doi.org/10.1038/s41594-018-0099-4
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DOI: https://doi.org/10.1038/s41594-018-0099-4
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