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Closing in on ATPase Activity by an RNA Helicase
Structure
Preview Closing in on ATPase Activity by an RNA Helicase Sean J. Johnson1,* and Matthew K. Yim1 1Department of Chemistry & Biochemistry, Utah State University, Logan, UH, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2020.01.007
In this issue of Structure, Absmeier et al. (2020) describe the molecular mechanisms employed by an RNA helicase to prevent premature ATP hydrolysis upon nucleotide binding. Helicases are nucleoside triphosphate (NTP)-driven enzymes that bind to and often unwind or remodel nucleic acid and ribonucleoprotein (RNP) substrates (Fairman-Williams et al., 2010). Helicases participate in nearly every biological process involving DNA and RNA. While diverse in structure and function, a common theme is the use of conserved sequence motifs located in two RecAlike domains to bind and hydrolyze nucleotides. These domains, named for their structural similarity to the RecA protein, are positioned adjacent to one another, forming a cleft that binds NTPs. NTP binding induces conformational rearrangements within and between the RecA domains that allow the helicase to perform its biological function. Importantly, the molecular details underlying active site rearrangement of NTPase activity differ between helicase families and in many cases are not well understood. Given the ability of RecA-like domains to catalyze NTP hydrolysis, one might ask how a helicase prevents unnecessary and wasteful NTPase activity, particularly in the absence of a DNA/RNA substrate. In this issue of Structure, a series of crystal structures provides an example of how intrinsic ATPase activity is inhibited in the RNA helicase Brr2 (Absmeier et al., 2020). Brr2 belongs to the Ski2-like family of superfamily 2 (SF2) helicases. The Ski2-like family is composed of helicases involved in a variety of biological processes including pre-mRNA splicing, RNA turnover, recombination, and DNA repair. Brr2 functions as a highly regulated component of the U5 small nuclear (sn)RNP spliceosomal complex and is involved in spliceosomal activation, splicing catalysis, and spliceosome disassembly. Ski2-like helicases all share a common core architecture containing four domains assembled in a ring-like structure (Johnson and Jackson, 2013). The first two do-
mains are RecA domains (RecA1 and RecA2) positioned adjacent to each other in sequence, followed by a winged helix (WH) and a helical bundle (HB) domain. Two additional domains in Brr2 (helixloop-helix [HLH] and immunoglobulinlike [IG] domains) complete the catalytically active ‘‘N-terminal cassette’’ (NC). Uniquely, the NC is followed by a second inactive ‘‘C-terminal cassette’’ (CC) with the same domain architecture. In addition, Brr2 contains a long (500 aa) N-terminal region (NTR) that inhibits ATPase, RNA binding, and unwinding activities by binding along one face of the NC, blocking the RNA binding site, and locking the RecA domains in an open conformation (Absmeier et al., 2015). In order to capture various nucleotide binding conformations in Brr2, Markus Wahl and colleagues used a ‘‘T4’’ truncation of Chaetomium thermophilum Brr2 (ctBrr2T4) where 441 residues were deleted from the NTR (Absmeier et al., 2020). The authors previously showed that truncation at the T4 position resulted in a significant increase in intrinsic ATPase activity, presumably as a result of ‘‘unlocking’’ the RecA domains (Absmeier et al., 2015). The new apo, ADP-bound, and ATPgS-bound structures of ctBrr2T4 highlight the influence of nucleotide binding at the interface of the RecA domains in the unlocked state (Figure 1). In the apo and ADP-bound structures, the RecA domains move closer together by approximately 2 A˚ as compared to the locked conformation. As expected, ADP binding engages residues in the conserved motifs Q and I of RecA1 and motif Va in RecA2. Binding of ATPgS (a non-hydrolyzable ATP analog) additionally engages residues in motif II of RecA1 and VI in RecA2, resulting in further closure of the RecA domains by another 2 A˚. Surprisingly, the ATPgS-bound active site adopts a previously unobserved
conformation that, while closed, is still not properly assembled for catalysis. Comparison of the ATPgS-bound structure to a catalytically competent ADP$BeF3-RNAbound structure of Prp43 (an SF2 RNA helicase from the DEAH/RHA-box family) (Tauchert et al., 2017) identifies important differences between the two active sites. The Brr2 active site is closed in a manner that excludes a water required for catalysis, thereby preventing ATP hydrolysis. It appears that RNA binding in the Prp43 structure induces additional conformational rearrangements to accommodate a water molecule, resulting in a catalytically competent active site. It is proposed that RNA binding to Brr2 would similarly produce a properly formed active site. Additional structures of Brr2 bound to RNA and nucleotides are needed to confirm this hypothesis. The structures determined by (Absmeier et al., 2020), along with previously determined structures, indicate that auto-inhibition of ATPase activity is achieved through both NTR binding and occlusion of a water molecule from the active site in the absence of RNA. In the cellular environment, unlocking of the RecA domains is accomplished by displacement of the NTR by other components of the spliceosome. The cryoelectron microscopy (cryo-EM) structure of the yeast U4/U6,U5 tri-snRNP spliceosome complex shows the NTR of Brr2 interacting with stem II of U4/U6 snRNA and a long helix of Prp3, thereby removing it from contact with the WH and HB domains and unlocking the RecA domains (Nguyen et al., 2016). The emerging story from each of these structural studies is that Brr2 ATPase activity is elegantly controlled through the combined influence of RNA substrates, protein-protein interactions, and accessory domains such as the NTR on RecA domain conformation (Figure 1).
Structure 28, February 4, 2020 ª 2020 Elsevier Ltd. 143
Structure
Preview
ACKNOWLEDGMENTS Are the auto-inhibitory mechanisms employed by We acknowledge the many excellent Brr2 utilized in other Ski2structural studies that could not be like helicases? The answer cited here due to space limitations. is unclear since comprehenSupport for this work was provided by National Institutes of Health sive nucleotide-bound strucR01GM117311. tural studies have not been reported for other Ski2-like REFERENCES helicases. Furthermore, the NTR of Brr2 is much longer Absmeier, E., Wollenhaupt, J., than that found in other Mozaffari-Jovin, S., Becke, C., Lee, C.T., Preussner, M., Heyd, F., Urlaub, Ski2-like helicases. Howev€hrmann, R., Santos, K.F., and H., Lu er, other accessory domains Wahl, M.C. (2015). The large may play a similar role. For N-terminal region of the Brr2 RNA helicase guides productive spliceosome example, a large insertion activation. Genes Dev. 29, 2576–2587. domain in the exosome-actiAbsmeier, E., Santos, K.F., and vating Ski2 helicase appears Wahl, M.C. (2020). Molecular to repress ATPase activity Mechanism Underlying Inhibition of in the yeast Ski complex Intrinsic ATPase Activity in a Ski2like RNA Helicase. Structure 28, (Halbach et al., 2013). More this issue, 236–243. broadly speaking, RNA/ DNA-stimulated ATPase acFairman-Williams, M.E., Guenther, U.P., and Jankowsky, E. (2010). SF1 tivity is a common theme and SF2 helicases: family matters. throughout the family, sugCurr. Opin. Struct. Biol. 20, 313–324. gesting that binding of Halbach, F., Rode, M., and Conti, E. the nucleic acid substrate (2012). The crystal structure of S. promotes conformational cerevisiae Ski2, a DExH helicase associated with the cytoplasmic changes within the RecA dofunctions of the exosome. RNA 18, mains to facilitate transition 124–134. from a pre-catalytic state to Halbach, F., Reichelt, P., Rode, M., Figure 1. Auto-inhibition of Brr2 ATPase Activity by the NTR and a fully active catalytic state. and Conti, E. (2013). The yeast ski Water Exclusion Modulation of ATPase and/ complex: crystal structure and RNA Truncation (T4) or displacement of the NTR (black) unlocks the RecA domains channeling to the exosome complex. or unwinding activity through (blue and red), allowing them to close upon ATP binding. However, Brr2 Cell 154, 814–826. protein-protein interactions is remains in a pre-catalytic state that excludes a required water molecule from the active site. It is proposed that RNA binding (as seen in the U4/U6$U5 Johnson, S.J., and Jackson, R.N. another common theme. Extri-snRNP structure) promotes transition to a catalytically competent state. (2013). Ski2-like RNA helicase strucamples include Brr2 (spliceoFor clarity, only the NTR, RecA, WH, and HB of the N-terminal cassette tures: common themes and complex some), Ski2 (Ski complex), are shown. assemblies. RNA Biol. 10, 33–43. and Mtr4 (TRAMP and NEXT Nguyen, T.H.D., Galej, W.P., Bai, complexes). X.C., Oubridge, C., Newman, A.J., To date, the observed 4 A˚ displace- ments are seen in other domains as a Scheres, S.H.W., and Nagai, K. (2016). Cryo-EM of the yeast U4/U6.U5 tri-snRNP at ment of the unlocked Brr2 RecA domains result of crystal contacts or protein-pro- structure 3.7 A˚ resolution. Nature 530, 298–302. upon ATPgS binding compared to the tein/protein-RNA interactions (Johnson NTR locked state is larger than any mo- and Jackson, 2013; Schuller et al., 2018; Schuller, J.M., Falk, S., Fromm, L., Hurt, E., and tions observed in multiple structures of Weick et al., 2018). If the RecA domains Conti, E. (2018). Structure of the nuclear exosome other Ski2-like family members. For of these helicases undergo structural re- captured on a maturing preribosome. Science 360, 219–222. example, while a structure of Ski2 bound arrangements similar to Brr2, it is not to the non-hydrolyzable ATP analog, evident from the existing structural data. Tauchert, M.J., Fourmann, J.B., Lu€hrmann, R., and AMPPNP, exhibits a possible pre-cata- As shown in this new work (Absmeier Ficner, R. (2017). Structural insights into the mechlytic state, the overall positioning of the et al., 2020), unlocking the Brr2 RecA anism of the DEAH-box RNA helicase Prp43. eLife 6, e21510. RecA domains does not change appre- domains enabled observation of RecA ciably when compared to apo-Ski2 (Hal- domain dynamics previously unobserved Weick, E.M., Puno, M.R., Januszyk, K., Zinder, bach et al., 2012, 2013). Similarly, multiple in Ski2-like family helicases. Perhaps J.C., DiMattia, M.A., and Lima, C.D. (2018). structures of Mtr4 reveal minimal con- there are restraining or locking mecha- Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA formational differences in the RecA nisms employed by other Ski2-like family Exosome-MTR4 Complex. Cell 173, 1663– domains, although significant rearrange- members that remain to be identified. 1677.e21, e1621.
144 Structure 28, February 4, 2020
Preview Closing in on ATPase Activity by an RNA Helicase Sean J. Johnson1,* and Matthew K. Yim1 1Department of Chemistry & Biochemistry, Utah State University, Logan, UH, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2020.01.007
In this issue of Structure, Absmeier et al. (2020) describe the molecular mechanisms employed by an RNA helicase to prevent premature ATP hydrolysis upon nucleotide binding. Helicases are nucleoside triphosphate (NTP)-driven enzymes that bind to and often unwind or remodel nucleic acid and ribonucleoprotein (RNP) substrates (Fairman-Williams et al., 2010). Helicases participate in nearly every biological process involving DNA and RNA. While diverse in structure and function, a common theme is the use of conserved sequence motifs located in two RecAlike domains to bind and hydrolyze nucleotides. These domains, named for their structural similarity to the RecA protein, are positioned adjacent to one another, forming a cleft that binds NTPs. NTP binding induces conformational rearrangements within and between the RecA domains that allow the helicase to perform its biological function. Importantly, the molecular details underlying active site rearrangement of NTPase activity differ between helicase families and in many cases are not well understood. Given the ability of RecA-like domains to catalyze NTP hydrolysis, one might ask how a helicase prevents unnecessary and wasteful NTPase activity, particularly in the absence of a DNA/RNA substrate. In this issue of Structure, a series of crystal structures provides an example of how intrinsic ATPase activity is inhibited in the RNA helicase Brr2 (Absmeier et al., 2020). Brr2 belongs to the Ski2-like family of superfamily 2 (SF2) helicases. The Ski2-like family is composed of helicases involved in a variety of biological processes including pre-mRNA splicing, RNA turnover, recombination, and DNA repair. Brr2 functions as a highly regulated component of the U5 small nuclear (sn)RNP spliceosomal complex and is involved in spliceosomal activation, splicing catalysis, and spliceosome disassembly. Ski2-like helicases all share a common core architecture containing four domains assembled in a ring-like structure (Johnson and Jackson, 2013). The first two do-
mains are RecA domains (RecA1 and RecA2) positioned adjacent to each other in sequence, followed by a winged helix (WH) and a helical bundle (HB) domain. Two additional domains in Brr2 (helixloop-helix [HLH] and immunoglobulinlike [IG] domains) complete the catalytically active ‘‘N-terminal cassette’’ (NC). Uniquely, the NC is followed by a second inactive ‘‘C-terminal cassette’’ (CC) with the same domain architecture. In addition, Brr2 contains a long (500 aa) N-terminal region (NTR) that inhibits ATPase, RNA binding, and unwinding activities by binding along one face of the NC, blocking the RNA binding site, and locking the RecA domains in an open conformation (Absmeier et al., 2015). In order to capture various nucleotide binding conformations in Brr2, Markus Wahl and colleagues used a ‘‘T4’’ truncation of Chaetomium thermophilum Brr2 (ctBrr2T4) where 441 residues were deleted from the NTR (Absmeier et al., 2020). The authors previously showed that truncation at the T4 position resulted in a significant increase in intrinsic ATPase activity, presumably as a result of ‘‘unlocking’’ the RecA domains (Absmeier et al., 2015). The new apo, ADP-bound, and ATPgS-bound structures of ctBrr2T4 highlight the influence of nucleotide binding at the interface of the RecA domains in the unlocked state (Figure 1). In the apo and ADP-bound structures, the RecA domains move closer together by approximately 2 A˚ as compared to the locked conformation. As expected, ADP binding engages residues in the conserved motifs Q and I of RecA1 and motif Va in RecA2. Binding of ATPgS (a non-hydrolyzable ATP analog) additionally engages residues in motif II of RecA1 and VI in RecA2, resulting in further closure of the RecA domains by another 2 A˚. Surprisingly, the ATPgS-bound active site adopts a previously unobserved
conformation that, while closed, is still not properly assembled for catalysis. Comparison of the ATPgS-bound structure to a catalytically competent ADP$BeF3-RNAbound structure of Prp43 (an SF2 RNA helicase from the DEAH/RHA-box family) (Tauchert et al., 2017) identifies important differences between the two active sites. The Brr2 active site is closed in a manner that excludes a water required for catalysis, thereby preventing ATP hydrolysis. It appears that RNA binding in the Prp43 structure induces additional conformational rearrangements to accommodate a water molecule, resulting in a catalytically competent active site. It is proposed that RNA binding to Brr2 would similarly produce a properly formed active site. Additional structures of Brr2 bound to RNA and nucleotides are needed to confirm this hypothesis. The structures determined by (Absmeier et al., 2020), along with previously determined structures, indicate that auto-inhibition of ATPase activity is achieved through both NTR binding and occlusion of a water molecule from the active site in the absence of RNA. In the cellular environment, unlocking of the RecA domains is accomplished by displacement of the NTR by other components of the spliceosome. The cryoelectron microscopy (cryo-EM) structure of the yeast U4/U6,U5 tri-snRNP spliceosome complex shows the NTR of Brr2 interacting with stem II of U4/U6 snRNA and a long helix of Prp3, thereby removing it from contact with the WH and HB domains and unlocking the RecA domains (Nguyen et al., 2016). The emerging story from each of these structural studies is that Brr2 ATPase activity is elegantly controlled through the combined influence of RNA substrates, protein-protein interactions, and accessory domains such as the NTR on RecA domain conformation (Figure 1).
Structure 28, February 4, 2020 ª 2020 Elsevier Ltd. 143
Structure
Preview
ACKNOWLEDGMENTS Are the auto-inhibitory mechanisms employed by We acknowledge the many excellent Brr2 utilized in other Ski2structural studies that could not be like helicases? The answer cited here due to space limitations. is unclear since comprehenSupport for this work was provided by National Institutes of Health sive nucleotide-bound strucR01GM117311. tural studies have not been reported for other Ski2-like REFERENCES helicases. Furthermore, the NTR of Brr2 is much longer Absmeier, E., Wollenhaupt, J., than that found in other Mozaffari-Jovin, S., Becke, C., Lee, C.T., Preussner, M., Heyd, F., Urlaub, Ski2-like helicases. Howev€hrmann, R., Santos, K.F., and H., Lu er, other accessory domains Wahl, M.C. (2015). The large may play a similar role. For N-terminal region of the Brr2 RNA helicase guides productive spliceosome example, a large insertion activation. Genes Dev. 29, 2576–2587. domain in the exosome-actiAbsmeier, E., Santos, K.F., and vating Ski2 helicase appears Wahl, M.C. (2020). Molecular to repress ATPase activity Mechanism Underlying Inhibition of in the yeast Ski complex Intrinsic ATPase Activity in a Ski2like RNA Helicase. Structure 28, (Halbach et al., 2013). More this issue, 236–243. broadly speaking, RNA/ DNA-stimulated ATPase acFairman-Williams, M.E., Guenther, U.P., and Jankowsky, E. (2010). SF1 tivity is a common theme and SF2 helicases: family matters. throughout the family, sugCurr. Opin. Struct. Biol. 20, 313–324. gesting that binding of Halbach, F., Rode, M., and Conti, E. the nucleic acid substrate (2012). The crystal structure of S. promotes conformational cerevisiae Ski2, a DExH helicase associated with the cytoplasmic changes within the RecA dofunctions of the exosome. RNA 18, mains to facilitate transition 124–134. from a pre-catalytic state to Halbach, F., Reichelt, P., Rode, M., Figure 1. Auto-inhibition of Brr2 ATPase Activity by the NTR and a fully active catalytic state. and Conti, E. (2013). The yeast ski Water Exclusion Modulation of ATPase and/ complex: crystal structure and RNA Truncation (T4) or displacement of the NTR (black) unlocks the RecA domains channeling to the exosome complex. or unwinding activity through (blue and red), allowing them to close upon ATP binding. However, Brr2 Cell 154, 814–826. protein-protein interactions is remains in a pre-catalytic state that excludes a required water molecule from the active site. It is proposed that RNA binding (as seen in the U4/U6$U5 Johnson, S.J., and Jackson, R.N. another common theme. Extri-snRNP structure) promotes transition to a catalytically competent state. (2013). Ski2-like RNA helicase strucamples include Brr2 (spliceoFor clarity, only the NTR, RecA, WH, and HB of the N-terminal cassette tures: common themes and complex some), Ski2 (Ski complex), are shown. assemblies. RNA Biol. 10, 33–43. and Mtr4 (TRAMP and NEXT Nguyen, T.H.D., Galej, W.P., Bai, complexes). X.C., Oubridge, C., Newman, A.J., To date, the observed 4 A˚ displace- ments are seen in other domains as a Scheres, S.H.W., and Nagai, K. (2016). Cryo-EM of the yeast U4/U6.U5 tri-snRNP at ment of the unlocked Brr2 RecA domains result of crystal contacts or protein-pro- structure 3.7 A˚ resolution. Nature 530, 298–302. upon ATPgS binding compared to the tein/protein-RNA interactions (Johnson NTR locked state is larger than any mo- and Jackson, 2013; Schuller et al., 2018; Schuller, J.M., Falk, S., Fromm, L., Hurt, E., and tions observed in multiple structures of Weick et al., 2018). If the RecA domains Conti, E. (2018). Structure of the nuclear exosome other Ski2-like family members. For of these helicases undergo structural re- captured on a maturing preribosome. Science 360, 219–222. example, while a structure of Ski2 bound arrangements similar to Brr2, it is not to the non-hydrolyzable ATP analog, evident from the existing structural data. Tauchert, M.J., Fourmann, J.B., Lu€hrmann, R., and AMPPNP, exhibits a possible pre-cata- As shown in this new work (Absmeier Ficner, R. (2017). Structural insights into the mechlytic state, the overall positioning of the et al., 2020), unlocking the Brr2 RecA anism of the DEAH-box RNA helicase Prp43. eLife 6, e21510. RecA domains does not change appre- domains enabled observation of RecA ciably when compared to apo-Ski2 (Hal- domain dynamics previously unobserved Weick, E.M., Puno, M.R., Januszyk, K., Zinder, bach et al., 2012, 2013). Similarly, multiple in Ski2-like family helicases. Perhaps J.C., DiMattia, M.A., and Lima, C.D. (2018). structures of Mtr4 reveal minimal con- there are restraining or locking mecha- Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA formational differences in the RecA nisms employed by other Ski2-like family Exosome-MTR4 Complex. Cell 173, 1663– domains, although significant rearrange- members that remain to be identified. 1677.e21, e1621.
144 Structure 28, February 4, 2020