Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase

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Lambowitz and co-workers address an important question in RNA biology: how RNA helicases have become specific for RNA, even though they are structurally very similar to DNA helicases. Using structural and biochemical approaches on the DEAD-box helicase Mss116 that functions as a chaperone for the process of mitochondrial intron splicing, they show that this helicase can, in principle, bind both RNA and DNA, and a variety of nucleotides, but with a significant preference for A over the others. Crystal structures are presented of the Mss116 construct in complex with Berylium Fluoride and ADP, CDP, GDP and UDP. These structures go some way to delineate specific interactions that confer specificity for the adenine base and RNA, and show that these specificities are caused by rather small tweaks in the helicase core. Their results also suggest that the gamma phosphate of ATP pays a critical role in maintaining a 'closed state' conformation when engaging the nucleotide and RNA. The authors also show that, in addition to the known ability to bind duplex RNA, Mss116 is capable of binding to A-form double stranded DNA, but not DNA in the more common B-form, while the isolated C-terminal RecA domain (D2) can bind the B-form. Crystal structures are also presented of Mss116 in complex with A10 RNA and A10 DNA, showing a similar mechanism of interaction for both substrates.

In general, the paper is well written and the methodology is sound, and the experimental work, encompassing X-ray crystallography and in vitro binding and unwinding assays, is comprehensive. The Discussion is instructive and insightful. The figures are excellent and are very easy on the eye. Although the results are perhaps not ground breaking, the paper provides new, and in some aspects surprising, insight for a large and ubiquitous family of enzymes with central biological roles. The manuscript will thus be of interest to a diverse readership. There are a number of minor comments on specific points that will hopefully be helpful for the authors to consider, outlined below.

1) Is it clear that Mss116 functions in vivo in isolation, or is the enzyme's activity directed and dependent upon other partner proteins? How might this bear on the central conclusion of the Abstract and Discussion, regarding the apparent family-specific specificity? Many of the DExD-box proteins seem to have partners that strongly affect their activity.

2) A surprising finding is the ability of Mss116 to unwind pure DNA duplexes, provided these adopt A-form geometry. The unwinding is shown with ADP-BeFx, a non-hydrolysable ATP analog. It would be important to check whether unwinding is also seen with ATP, as several recent papers suggest that DEAD-box helicases, which have previously been considered as pure RNA helicases, might function on DNA.

3) Unwinding of RNA with all four nucleotide diphosphate beryllium fluoride compounds is also surprising. Here, again, it would be instructive to test unwinding with the nucleotide triphosphates. These results might be influenced by the affinity of binding to the nucleotide rather than the ability of the helicase to hydrolyse a particular nucleotide and simultaneously unwind the duplex, for example. In addition, the different triphosphates have been tested in unwinding assays for many DEAD-box helicases, and it appears that no unwinding has been reported with NTPs other than ATP. It would thus be important to sort out whether one should have tested these NTPs over a larger concentration range than examined, or whether the use of the diphosphate beryllium fluoride compounds somehow biases these experiments, as has been suggested, too. If a bias were to be found, this would not invalidate any of the points made, but add a further important facet to the story.

4) The authors report K_d values for ADP-BeFx binding to Mss116 in the micromolar range. Binding of ADP-BeFx to Mss116 and RNA has recently been described by Liu et al. (Biochemistry, 2014, 53, 423–433). Although no K_d values were reported there, the ADP-BeFx complex with Mss116 was very long-lived. It might be prudent to specifically verify that equilibrium was reached for the K_d measurements, even after incubation of 1h. In addition, the paper by Liu et al. should probably be cited.

5) In Figure 1D it might be helpful to extend the cartoon to include the final step in the unwinding cycle where the ATP is hydrolysed, and then the helicase re-enters the open state upon departure of the ADP, Pi and RNA.

6) In Figure 2, perhaps it would be better to have the binding assay (Figure B) first, then the unwinding assay (A).

7) Figure 3: Label G128 in Figure B? Is the Q133 oriented such that the carbonyl is forced to be pointed at the O6 of the GDP? This would be an unfavorable interaction.

8) Results section, “The structural basis for the ATP specificity of the helicase core of Mss116”: “two side-chain hydrogen (H)-bonds from G128 and E133”. Figure 3 has residue 133 labelled as Q. Which one is it? Also, can glycine really make a side chain H-bond?

9) Summary: 'core stability'. Perhaps change the wording here, because as written this might be misinterpreted as stability of the fold, which is clearly not intended.

10) Results section (“analytical size-exclusion chromatography (SEC) shows that a 94 closed-state helicase…”), Table 1 and Figure 5–figure supplement 2: do the elution volumes definitively demonstrate whether the state is open or closed? There is for example the formal possibility that the proteins are forming oligomers. Do the authors have access to SEC-MALS? This would give the molecular weights fairly precisely, which can be used to confirm that the complexes are intact.

11) Results section: the sentence “the effective concentration of the ATP gamma phosphate plays a key role in maintaining the closed-state structure” appears to be repetitive with “the effective concentration of the ATP gamma phosphate is critical for the stability of the closed-state”.

12) Table 2: the angles for the unit cell are not in Greek characters.

13) Figure 2–figure supplement 1 appears to be missing.

14) Figure 4E: out of curiosity, what happens to the binding of the A-form DNA with D2, does it retain A-form CD spectral signature?

1) Is it clear that Mss116 functions in vivo in isolation, or is the enzyme's activity directed and dependent upon other partner proteins? How might this bear on the central conclusion of the Abstract and Discussion, regarding the apparent family-specific specificity? Many of the DExD-box proteins seem to have partners that strongly affect their activity.

Mss116 by itself displays high unwinding activity in the absence of partner proteins in vitro, and functional studies give no indication that Mss116’s helicase activity is directed by or dependent upon a partner protein in vivo. Instead, Mss116 functions as a general RNA chaperone that binds diverse RNA and RNP substrates non-specifically and resolves kinetic traps that impede RNA folding (Huang et al., 2005; Del Campo et al., 2009). In vivo, Mss116 is required for the efficient splicing of all 13 yeast mitochondrial group I and group II introns, as well as the translation of certain mRNAs and mitochondrial RNA processing reactions (Huang et al., 2005). The introns whose splicing is promoted by Mss116 differ structurally and rely on different intron-encoded maturases or nuclear gene-encoded splicing factors for structural stabilization. In vitro, purified Mss116 can by itself promote splicing of group II introns or function in the presence of a heterologous splicing factor, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein), to promote the splicing of a N. crassa group I intron (Del Campo et al., 2009). The lack of specificity required for Mss116 function in vivo is further indicated by the finding that all phenotypic defects in an mss116Δ strains can be rescued by the expression of the N. crassa CYT-19 protein (Huang et al., 2005), a related DEAD-box that also functions as general RNA chaperone (Mohr et al., 2002).

In addition to its function as a general RNA chaperone, Mss116 co-purifies with the yeast mt RNA polymerase (Markov et al., 2009) and may function as a transcription elongation factor for this enzyme (Markov et al., 2014). However, this function does not require the helicase activity (i.e., the ATPase or RNA-unwinding activities) of Mss116.

To address this comment, we have modified the Introduction to state that Mss116 binds diverse RNA substrates non-specifically and displays high RNA helicase activity in the absence of partner proteins.

2) A surprising finding is the ability of Mss116 to unwind pure DNA duplexes, provided these adopt A-form geometry. The unwinding is shown with ADP-BeFx, a non-hydrolysable ATP analog. It would be important to check whether unwinding is also seen with ATP, as several recent papers suggest that DEAD-box helicases, which have previously been considered as pure RNA helicases, might function on DNA.

We performed additional kinetic unwinding assays, which demonstrate that Mss116 can use ATP to unwind the RNA duplex and the A-DNA duplex substrates with observed first-order rate constants (k₁) of 0.46 and 0.15 min^-1, respectively. However, we do not observe unwinding of the B-DNA duplex with ATP. This is the same unwinding trend that we observed with ADP-BeF_x. The kinetic unwinding assays have been added to the Results (Figure 5– figure supplement 3) and to the Methods sections, along with references to recent papers suggesting DEAD-box helicases might function on DNA (Kammel et al., 2013; Tuteja et al., 2014).

3) Unwinding of RNA with all four nucleotide diphosphate beryllium fluoride compounds is also surprising. Here, again, it would be instructive to test unwinding with the nucleotide triphosphates. These results might be influenced by the affinity of binding to the nucleotide, rather than the ability of the helicase to hydrolyse a particular nucleotide and simultaneously unwind the duplex, for example. In addition, the different triphosphates have been tested in unwinding assays for many DEAD-box helicases, and it appears that no unwinding has been reported with NTPs other than ATP. It would thus be important to sort out whether one should have tested these NTPs over a larger concentration range than examined, or whether the use of the diphosphate beryllium fluoride compounds somehow biases these experiments, as has been suggested, too. If a bias were to be found, this would not invalidate any of the points made, but add a further important facet to the story.

We performed kinetic unwinding assays, as in point 2, to compare the unwinding of the 12-bp RNA duplex catalyzed by ATP to CTP, GTP and UTP, which are documented in the Methods and reported in the Results (Figure 2–figure supplement 2). These assays demonstrate that NTPs other than ATP do not promote unwinding of the RNA duplex even with the other NTPs added at 5 mM concentration. We also performed these kinetic unwinding assays in a different buffer with 0.5 mM free Mg²⁺, as previous data indicates that the unwinding activity of Mss116 increases at lower Mg²⁺ concentrations (Halls et al., 2007). However, we still found no unwinding for CTP, GTP or UTP under these conditions. These findings suggest that the closed state with ssRNA and nucleotide triphosphates other than ATP does not form and catalyse the unwinding of the RNA duplex used in these assays. Use of the diphosphate beryllium fluoride analogues is therefore necessary to access and stabilize the closed-states that promote unwinding with other nucleotide bases.

4) The authors report K_d values for ADP-BeFx binding to Mss116 in the micromolar range. Binding of ADP-BeF_x to Mss116 and RNA has recently been described by Liu et al. (Biochemistry, 2014, 53, 423–433). Although no K_d values were reported there, the ADP-BeF_x complex with Mss116 was very long-lived. It might be prudent to specifically verify that equilibrium was reached for the K_d measurements, even after incubation of 1h. In addition, the paper by Liu et al. should probably be cited.

We had verified that equilibrium was reached in all our experiments by carrying out assays for extend times (up to approximately 4 h), which gave the same binding or unwinding profiles as those incubated for 1 h. This information has been incorporated into the Methods section. We note that the protein (full-length Mss116 in Liu et al. versus the helicase core in our study), RNA substrates, and reaction conditions differ between the two studies. We have cited the paper by Liu et al. as example of the different behaviors of ATP analogues.

5) In Figure 1D it might be helpful to extend the cartoon to include the final step in the unwinding cycle where the ATP is hydrolysed, and then the helicase re-enters the open state upon departure of the ADP, P_i and RNA.

We have added this step final step in the unwinding cycle to Figure 1D.

6) In Figure 2, perhaps it would be better to have the binding assay (Figure B) first, then the unwinding assay (A).

In the current version of the manuscript, the panels that display the unwinding (Figure 2A) and binding (Figure 2B) appear in the figure in the order that they are mentioned in the text.

7) Figure 3: Label G128 in Figure B? Is the Q133 oriented such that the carbonyl is forced to be pointed at the O6 of the GDP? This would be an unfavorable interaction.

The O-O distance from the carbonyl of the Q133 side chain and the O6 of the GDP is 3.4 Å in the GDP structure. This is compared to 2.4 Å in the ADP structure, even though the two distances look similar in the view in Figure 3B. The carbonyl of Q133 is therefore not pointing directly towards the O6 of the GDP, and the only favorable contact with this base is made by Q133 to N7.

8) Results section, “The structural basis for the ATP specificity of the helicase core of Mss116”: “two side-chain hydrogen (H)-bonds from G128 and E133”. Figure 3 has residue 133 labelled as Q. Which one is it? Also, can glycine really make a side chain H-bond?

We have corrected this sentence to read: ‘two hydrogen (H)-bonds from G128 and Q133…”.

9) Summary: 'core stability'. Perhaps change the wording here, because as written this might be misinterpreted as stability of the fold, which is clearly not intended.

We have changed ‘core stability’ to ‘complex stability’.

10) Results section (“analytical size-exclusion chromatography (SEC) shows that a 94 closed-state helicase…”), Table1 and Figure 5–figure supplement 2: do the elution volumes definitively demonstrate whether the state is open or closed? There is for example the formal possibility that the proteins are forming oligomers. Do the authors have access to SEC-MALS? This would give the molecular weights fairly precisely, which can be used to confirm that the complexes are intact.

The ratio of A₂₆₀/A₂₈₀, which is approximately 0.6 for free protein and >1 for protein-nucleic acid complexes, was used as an indicator of the formation of a closed-state complex that contains nucleotide and nucleic acid. Protein cores that remain mostly in the open state under the SEC conditions have a A₂₆₀/A₂₈₀ ratio much closer to that of the free protein. The elution volumes are consistent with the complex formation and are used only in conjunction with the A₂₆₀/A₂₈₀ ratio as indicating the open or closed state of the core.

11) Results section: the sentence “the effective concentration of the ATP gamma phosphate plays a key role in maintaining the closed-state structure” appears to be repetitive with “the effective concentration of the ATP gamma phosphate is critical for the stability of the closed-state”.

We have removed the sentence: “They also indicate that the effective concentration of the ATP gamma phosphate plays a key role in maintaining the closed-state structure”.

12) Table 2: the angles for the unit cell are not in Greek characters.

We have changed the angles of the unit cell to Greek characters.

13) Figure 2–figure supplement 1 appears to be missing.

Apologies for the omission of this Figure supplement, it is now included.

14) Figure 4E: out of curiosity, what happens to the binding of the A-form DNA with D2, does it retain A-form CD spectral signature?

We have now also measured the CD spectra of the A-form DNA duplex when bound to Mss116 D2, and this duplex also retains A-form geometry upon binding. This is included as Figure 4–figure supplement 1.