Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP Into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1

doi:10.1016/j.jmb.2009.12.025 J. Mol. Biol. (2010) 396, 949–966 Available online at www.sciencedirect.com Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1 Josette Banroques1,2,3, Monique Doère3, Marc Dreyfus1, Patrick Linder3 and N. Kyle Tanner1,3⁎ 1Institut de Biologie Motif III in the putative helicases of superfamily 2 is highly conserved in Physico-chimique, CNRS UPR both its sequence and its structural context. It typically consists of the 9073 in association with the sequence alcohol–alanine–alcohol (S/T-A-S/T). Historically, it was thought Université Paris VII, Paris to link ATPase activity with a “helicase” strand displacement activity that 75005, France disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP- 2Centre de Génétique dependent RNA binding proteins that, in some cases, are able to disrupt Moléculaire, CNRS FRE 3144, short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast Gif-sur-Yvette 91198, France DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro 3Département de Microbiologie properties. Moreover, we made a tertiary model of Ded1 based on the et Médecine Moléculaire, Centre solved structure of Vasa. We used Ded1 because it has relatively high Médical Universitaire, Geneva ATPase and RNA binding activities; it is able to displace moderately stable 1211, Switzerland duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important Received 6 August 2009; than the serine, but that mutations of all three residues have strong received in revised form phenotypes. We purified the wild-type and various mutants expressed in 8 December 2009; Escherichia coli. We found that motif III mutations affect the RNA-dependent accepted 14 December 2009 hydrolysis of ATP (kcat), but not the affinity for ATP (Km). Moreover, Available online mutations alter and reduce the affinity for single-stranded RNA and 21 December 2009 subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the γ binding energy of -PO4 of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP. © 2009 Elsevier Ltd. All rights reserved. Keywords: RNA helicase; ATPase; molecular motor; Saccharomyces cerevisiae; Edited by A. Pyle RecA like Introduction The putative helicases of superfamily 2 (SF2) are an ubiquitous group of enzymes that are associated *Corresponding author. IBPC, CNRS UPR Pierre et Marie with all processes involving RNA and DNA. SF2 Curie, 75005 Paris, France. E-mail address: proteins are closely related to those of superfamily 1 [email protected]. (SF1), some of which are known processive DNA Abbreviations used: SF2, superfamily 2; SF1, helicases that are involved in DNA replication and 1,2 superfamily 1; ssRNA, single-stranded RNA; AMP-PNP, repair. These NTPases (generally ATPases) are adenosine 5′-(β,γ-imino)triphosphate; PDB, Protein Data characterized by a highly conserved structural core Bank; SD-Leu plate, synthetic minimal medium plate consisting of two linked RecA-like domains that lacking leucine; 5-FOA, 5-fluoroorotic acid; EMSA, contain seven or more conserved motifs involved in electrophoretic mobility shift assay. nucleotide triphosphate and nucleic acid binding, 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved. 950 Motif III in Superfamily 2 “Helicases” interdomain interactions, and NTPase activity.1,2 an essential gene in the yeast Saccharomyces cerevi- The largest SF2 family comprises DEAD-box siae. It was first identified as an intragenic suppres- proteins, which are RNA-dependent ATPases and sor of a PRP8 mutant, which implied a role in ATP-dependent RNA binding proteins. These mRNA splicing,22 but it was also implicated in the proteins are associated with all processes involving transcription of polymerase III RNAs,23 and to have RNA from transcription to decay, and each family a general role in translation initiation24,25 and 40S member is typically involved in a unique ribosome scanning.26 Finally, it was implicated in P- – process.3 7 They contain nine conserved motifs body formation and RNA degradation27 and in (Q, I, Ia, Ib, and II–VI), a conserved GG sequence yeast L-A virus synthesis.28 Ded1 is closely related between motifs Ia and Ib, and a conserved QxxR to a subfamily of DEAD-box proteins involved in sequence (where x is any residue) between motifs developmental regulation,29–31 including the Dro- IV and V.3,4,7,8 Although commonly known as sophila Vasa protein for which the crystal structure DEAD-box “helicases,” they have not been shown was solved in the presence of RNA and adenosine to have a processive unwinding activity of double- 5′-(β,γ-imino)triphosphate (AMP-PNP).12 stranded RNA, and they are only able to displace Motif III is defined by the sequence serine– short duplexes, typically at very high protein/ alanine–threonine (S-A-T) in the DEAD-box family. duplex ratios in vitro.1,7,9 We mutated the three positions in DED1 and There are now a large number of solved crystal analyzed the in vivo phenotypes and in vitro structures of DEAD-box proteins, including several properties of the proteins expressed in and – with bound ligands.10 15 These data have verified or purified from Escherichia coli. We find that muta- clarified the roles of the different conserved motifs tions of motif III are poorly tolerated in vivo, and and features. Thus, the Q motif is involved in that these mutants affect the hydrolysis of ATP adenine recognition. Motifs I and II, also called (kcat) and the affinity for ssRNA, but not the Walker motifs A and B, are involved in the binding affinity for ATP (Km). This subsequently reduces of the α and β phosphates of the ATP. Motifs Ia, Ib, the ability of the proteins to displace nucleic acid IV, and V; the conserved sequences GG and QxxR; duplexes. It appears that motif III is critical for and a residue next to motif II are involved in single- aligning—and perhaps activating—the γ phos- stranded RNA (ssRNA) binding. Motifs III, V, and phate of ATP, for coordinating the different motifs, VI are involved in binding the α, β,andγ and for helping to convert the binding energy of phosphates of ATP.2,4,7,8 ATP into a high-affinity RNA binding site. Thus, The enzymatic roles of specific residues within the mutations of motif III in Ded1 affect the observed motifs of DEAD-box proteins were derived from “helicase” activity indirectly by altering and mutagenic and biochemical analyses. The highly reducing RNA binding, and this may be true for conserved glutamic acid of motif II, which is other characterized SF2 proteins as well. conserved in other SF2 and SF1 families as well, is β γ thought to activate the , -phosphoanhydride bond Results of ATP through a coordinated water molecule, in association with a conserved histidine residue of motif VI and a bound magnesium ion.4,16 A similar Sequence alignments in and around motif III mechanism of activation was proposed for the SF2 Dengue NS3 protein.17 The role of motif III is more Sequence alignments of 699 unique DEAD-box complicated; it has been suggested to link ATPase proteins were performed as described in Materials and “helicase” activities because mutations in and Methods and are shown in Fig. 1a. Motif III mammalian eIF4A eliminated strand displacement contained a serine in the first position 89% of the activity without affecting ATP hydrolysis or RNA time, while a threonine occurred 11% of the time. binding.18 In contrast, similar mutations in yeast The second position of alanine was 100% conserved, Has1 have reduced affinities for ATP and RNA and and there was virtually always a threonine in the reduced ATPase activity, which resulted in reduced last position, although a serine was found in 0.4% of strand displacement activity.5 Mutations of motif III the sequences. In contrast, the sequences flanking also dissociated the ATPase activity from the strand motif III were more variable, but there was a displacement activity in the related yeast DEAH-box tendency for hydrophobic residues to precede and, protein Prp22, but the RNA binding affinities were to a lesser extent, follow motif III. We examined the not determined.19 Finally, motif III was proposed to solved crystal structures of Vasa, DDX19B (human serve as a relay for ATP binding and hydrolysis and Dbp5), Mss116, and eIF4AIII in the presence of for the binding of DNA in the SF1 Rep protein, but bound RNA and AMP-PNP to better understand its – the character of motif III and its interactions with the structural context;10 15 motif III occurred at the end other motifs in SF1 are significantly different from of a β-sheet strand that subsequently turned into a those of SF2.20,21 loop, and this was probably why there was a We were interested in obtaining mutants of motif preference for hydrophobic groups preceding the III in the DEAD-box protein Ded1 that dissociated motif (Fig. 1a). The loop structure then turned into a the different activities, so that we could better helix, but there were differences between Vasa, understand the interrelationships between the DDX19B, Mss116, and the different eIF4AIII crystal motifs and the role of the protein in vivo.

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