Dual role of the RNA in selectivity and by terminal uridylyl

Jason Stagno†, Inna Aphasizheva‡, Ruslan Aphasizhev‡§, and Hartmut Luecke†§¶ʈ††

Departments of †Molecular Biology and Biochemistry, ‡Microbiology and Molecular Genetics, ¶Physiology and Biophysics, and ʈInformatics and Computer Science and ††Center for Biomembrane Systems, University of California, Irvine, CA 92697

Edited by David R. Davies, National Institutes of Health, Bethesda, MD, and approved July 18, 2007 (received for review May 11, 2007) Terminal RNA uridylyltransferases (TUTases) catalyze template- several novel TUTases, also referred to as poly(U) , independent UMP addition to the 3؅ hydroxyl of RNA. TUTases belong encoded in the human, Caenorhabditis elegans, and Arabidopsis to the DNA ␤ superfamily of that thaliana genomes (13). share a conserved catalytic domain bearing three metal-binding Recent crystallographic studies of TbRET2 (14) and TbTUT4 carboxylate residues. We have previously determined crystal struc- (8) attributed the specificity of UTP incorporation by trypanosomal tures of the UTP-bound and apo forms of the minimal trypanosomal TUTases to the largely conserved, high-affinity formed TUTase, TbTUT4, which is composed solely of the N-terminal catalytic at the interface of the N-terminal domain, which bears a polymer- and C-terminal base-recognition domains. Here we report crystal ase ␤ signature sequence with three catalytic aspartate residues, and structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating the C-terminal domain, which resembles an ATP cone-like fold also nearly perfect superposition of the triphosphate moieties with that of found in 2Ј–5Ј oligoadenylate synthetases (15) and is responsible for the UTP substrate. Consequently, at physiological nucleoside 5؅- base-specific contacts. By combining kinetic analysis and UTP triphosphate concentrations, the protein–uracil base interactions cross-linking studies of mutant TbTUT4 proteins, we have estab- alone are not sufficient to confer UTP selectivity. To resolve this lished the importance of the uracil base interactions for UTP ambiguity, we determined the crystal structure of a prereaction binding (8). Whereas the stacking interaction most likely contrib- ternary complex composed of UTP, TbTUT4, and UMP, which mimics utes to the selectivity of TbTUT4 toward pyrimidines, base-specific an RNA substrate, and the postreaction complex of TbTUT4 with UpU recognition of UTP is apparently achieved by endocyclic N3 dinucleotide. The UMP pyrimidine ring stacks against the uracil base donating a hydrogen bond to one water molecule and carbonyl O4 of the bound UTP, which on its other face also stacks with an essential receiving a hydrogen bond from another water molecule, which tyrosine. In contrast, the different orientation of the purine bases would require reversal of this hydrogen-bonding pattern at these observed in cocrystals with ATP and GTP prevents this triple stacking, two positions for effective CTP binding (8). precluding productive binding of the RNA. The 3؅ hydroxyl of the The spacious UTP binding site formed at the interface of the bound UMP is poised for in-line nucleophilic attack while contributing N-terminal and C-terminal domains, remarkable sequence similar- to the formation of a binding site for a second catalytic metal ion. We ity between the catalytic domains of TUTases and noncanonical propose a dual role for RNA substrates in TUTase-catalyzed reactions: PAPs, and conservation of key residues involved in UTP binding contribution to selective incorporation of the cognate nucleoside and [supporting information (SI) Fig. 7] (1, 16) prompted us to inves- shaping of the catalytic metal binding site. tigate whether UTP–protein contacts alone are sufficient to confer U-specificity at nucleoside 5Ј-triphosphate (NTP) concentrations crystal structure ͉ nucleotidyl ͉ RNA editing ͉ Trypanosoma ͉ approaching physiological levels. Surprisingly, we were able to terminal RNA uridylyltransferase poly(A) polymerase generate cocrystals of TbTUT4 with CTP, ATP, and GTP under the same conditions used for TbTUT4:UTP binary complex crys- erminal RNA uridylyltransferases (TUTases) are phylogeneti- tallization (8). The x-ray structures of these complexes demon- Tcally widespread and functionally divergent that cat- strated nearly perfect superposition of the triphosphate moieties alyze template-independent transfer of UMP residues to the 3Ј while revealing a major reduction in the observed coplanarity of hydroxyl group of RNA (1). TUTases belong to the polymerase ␤ purine bases with respect to the phenyl ring of conserved Y189, superfamily, which is characterized by the relative to that of pyrimidines. This shift of base positioning presence of the signature motif hG[G/S]X9-13Dh[D/E]h (X, any; h, diminishes the stacking interaction with Y189 but does not fully hydrophobic amino acids) (2) and also includes poly(A) poly- explain the ’s ability to discriminate against purine NTPs. merases (PAPs), ATP(CTP):tRNA nucleotidyltransferases, termi- To further examine this phenomenon, we have established that nal deoxy nucleotidyltransferases, protein nucleotidyltransferases, TbTUT4 can use uridylyl monophosphate in lieu of an RNA primer 2Ј–5Ј oligo(A) synthetases, and antibiotic resistance nucleotidyl- and elucidated structures of a TbTUT4:UTP:UMP ternary com- transferases (3). TUTase activities have been reported in mamma- plex (precatalysis), as well as the structure of TbTUT4 with the Ј lian cells, plants, and parasitic protists from the order Kinetoplas- bound adduct, UpU (postcatalysis, but lacking the 5 phosphate). tida, Trypanosoma brucei, and Leishmania ssp. (1). Extensive uridine insertion/deletion editing in mitochondria of trypanosoma- Ј Author contributions: R.A. and H.L. designed research; J.S., I.A., R.A., and H.L. performed tids requires 3 uridylylation of guide by RNA editing research; I.A. contributed new reagents/analytic tools; J.S., I.A., R.A., and H.L. analyzed TUTase 1 (RET1) (4) and insertion of Us into messenger RNAs by data; and J.S., R.A., and H.L. wrote the paper. RNA editing TUTase 2 (RET2) (5, 6). In addition to mitochondrial The authors declare no conflict of interest. RNA editing TUTases, TUT3 (7) and TUT4 (8), cytoplasmic This article is a PNAS Direct Submission. uridylyl transferases have been reported in T. brucei. Human cells Abbreviations: TUTase, terminal RNA uridylyltransferase; NTP, nucleoside 5Ј-triphosphate; apparently possess several distinct TUTase activities, of which the PAP, poly(A) polymerase; PDB, Protein Data Bank. U6 small nuclear RNA-specific TUTase (9) and a UTP-specific Data deposition: The atomic coordinates and structure factors have been deposited in the enzyme of unknown function (10) have been identified. Cid1, a Protein Data Bank, www.pdb.org (PDB ID codes 2IKF, 2B51, and 2B56). member of a multifunctional in fission yeast, was §To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. characterized as a cytoplasmic PAP (11) that also possesses a robust This article contains supporting information online at www.pnas.org/cgi/content/full/ TUTase activity (10). An enzymatic activity screening of proteins 0704259104/DC1. homologous to the animal Gld-2 cytoplasmic PAP (12) revealed © 2007 by The National Academy of Sciences of the USA

14634–14639 ͉ PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704259104 Downloaded by guest on September 28, 2021 Table 1. Kinetic constants for NTP incorporation by TbTUT4 and GTP. Kinetic parameters of the TbTUT4-catalyzed reac- tion in the presence of different ribonucleoside triphosphates kcat/Km, ͓kcat/Km͔UTP/ Ϫ1 Ϫ1 Ϫ1 demonstrated that non-UTP substrates are characterized by NTP Km, ␮M kcat, min min ⅐M ͓kcat/Km͔NTP similar values for the apparent Km, which are Ϸ20-fold higher UTP 1 Ϯ 0.9 0.5 Ϯ 0.06 5 ϫ 105 1.0 than that observed for the cognate substrate (Table 1). Sur- ATP 47.5 Ϯ 19.8 0.01 Ϯ 0.001 2 ϫ 102 0.0004 prisingly, the catalytic rate remained nearly identical for Ϯ Ϯ ϫ 4 CTP 17.1 2.6 0.5 0.03 2.9 10 0.06 pyrimidines while decreasing significantly for purines. This GTP 18.0 Ϯ 1.8 0.004 Ϯ 0.002 5 ϫ 102 0.0001 finding indicated that, at physiological concentrations of NTPs Data for UTP are from ref. 8. Reactions were performed with 6͓U͔ RNA as in vivo (Ͼ0.5 mM), UMP incorporation by TUTases may the substrate. require, in addition to specific UTP–protein contacts (8, 14), a supplementary component ensuring a selective reaction. The structures of TbTUT4 cocrystals with UTP, CTP, ATP, and We discovered that the terminal base of the RNA substrate forms GTP demonstrated nearly identical binding of the triphos- a ‘‘triple-stacked sandwich’’ with the bound UTP base and the phate moiety. However, deviations become evident in the Ј phenol ring of Y189, which positions its 3 hydroxyl group for in-line positioning of the ribose sugar pucker and, more prominently, ␣ attack on the -phosphorus atom of the UTP. In contrast, the the degree of coplanarity of the pyrimidine vs. purine bases observed positions of purine bases in ATP- and GTP-bound with respect to the phenyl ring of Y189 (Fig. 1). Detailed structures display reduced stacking interactions and likely destabi- analysis of the various TbTUT4:NTP cocrystal structures lize RNA binding. In addition, the 3Ј hydroxyl group of UMP illustrates four primary differences in the binding of purines (mimicking the 3Ј residue of the RNA substrate) completes a relative to that of pyrimidines: (i) a second metal ion of binding site for a second catalytic metal ion, which is typically unknown function is present in ATP and GTP complex required for the nucleoside transfer reaction (17) but has not been structures; (ii) N147, which forms hydrogen bonds with the 2Ј observed in previously reported TbTUT4:UTP and TbRET2:UTP hydroxyl group of the ribose and a carbonyl oxygen of the binary complexes [Protein Data Bank (PDB) ID codes 2IKF and pyrimidine bases, is involved in hydrogen bonding with both 2B56, respectively] (8, 14). Modeling of additional RNA residues ribose hydroxyl groups in the purine-bound structures; (iii)a into the ternary complex provides a rationale for the pronounced hydrogen bond is donated from the hydroxyl group of residue selectivity of trypanosomal TUTases toward the specific base at the T187 to N7 of the purine rings of ATP and GTP, whereas this 3Ј end of the RNA substrate. Thus, our studies establish a structural residue appears to play no role in pyrimidine binding; and (iv) model for UTP/ATP recognition by a conserved catalytic module the pose of the purine rings results in substantially reduced shared among RNA uridylyltransferases and noncanonical PAPs, a ␲-electron stacking with the aromatic ring of Y189, an inter- group of enzymes involved in a wide variety of RNA processing action previously shown to be essential for catalysis (8). These events in eukaryotes (1, 16). observations provide a structural explanation for the fact that enzyme–heterocyclic base contacts alone are not sufficient to Results confer UTP selectivity. This is consistent with the conserva- Nucleoside Triphosphate Binding by TbTUT4. Although TbTUT4 tion of the catalytic/base recognition bidomain scaffold among greatly favors UTP as its substrate, it has been shown that the TUTases and several divergent PAPs, such as trypanosomal enzyme is also capable of using, to various extents, CTP, ATP, mitochondrial PAP (R. D. Etheridge, I.A., and R.A., unpub-

A PTU PTC PTA PTG

631D D136 631D 631D

D 66 66D 66D 66D D68 86D 86D 86D

2gM 2gM gM 1 1gM gM 1 M 1g

S 56 S 56 56S S65 71K 3 K 371 371K S188 371K 881S S 81 8 881S 1K 96 961K 961K 961K

B 1S 84 1S 48 841S 1S 84

741N N1 74 741N N 741 81Y 9 1Y 98 Y 81 9 Y 81 9 792D 792D 792D 792D

81T 7 T 81 7 81T 7 781T BIOCHEMISTRY R 703 03R 7 703R 703R Fig. 1. Primary interactions in NTP binding by TbTUT4. Ordered water molecules and Mg2ϩ ions are depicted in C cyan and black, respectively. (A) The triphosphate moiety contacts. (B) Sugar/base interactions. (C) The relative po- sitions of all four bound triphosphate ribonucleosides TU P PTC PTA PTG derived from superpositioning the structures of TbTUT4:UTP, TbTUT4:CTP, TbTUT4:ATP, and TbTUT4:GTP. Composite annealed omit maps for each bound ligand observed in the various ligand complexes of TbTUT4 are shown in SI Fig. 8.

Stagno et al. PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 ͉ 14635 Downloaded by guest on September 28, 2021 Fig. 2. RNA substrate specificity of TbTUT4. (A) Dou- Rsd NA Tb UT 4T ble-stranded RNA substrates for precleaved in vitro A B bT bT [6 U] ]A[6 5 A]U[ insertion assays (4) were assembled before reaction TUT 4 2TER from the labeled 5Ј fragment, 3Ј fragment, and guide RNA, which allows for insertion of three, two, and zero . RNAs (0.5 ␮M) were incubated with 50 nM recombinant TbTUT4 or TbRET2 in the presence of 100 ␮M UTP for 30 min. Lane 1, control RNA; lane 2, 5Ј fragment alone; lane 3, 5Ј fragment hybridized with a ‘‘bridge’’; lanes 4, 5, and 6, fully assembled substrates with a 0-, 2-, or 3-nt gap. Products were separated on 1 2 3 4 5 6 15% polyacrylamide/8 M urea gels. (B) Single-stranded 1 2 3 4 5 6 5Ј radiolabeled RNA substrates ending with six Us, six 5´ rf ga em tn 3´ rf a mg ent As, or terminal A after five Us were incubated with 50 *5´ 3´ 5´ 3´ | | | | | | | | | | | | | | | | | c c c 5´ nM purified TbTUT4 in the presence of 1, 10, 100, and 3´ aga PTA PTU PTA PTU TA P PTU ANR ediug ANR ag 500 ␮M UTP for 30 min. c, control RNA. (C) TbTUT4 0 transfers uridylyl residues to UMP and UpU acting as D ANR-4TUT[ANR+]PTU-4TUT[ ] PTU+ 4TUT+]PTU-ANR[ RNA primers. In gels 1–3, the concentration of the 5Ј C Ј Ј 32 T[ UT4- pUp ]U [T TU 4 U- TP] [ p-PTU pU U] U[ PT - ]PMU radiolabeled uridylyl-3 ,5 -uridine ( pUpU) was kept TU+ P Up+ Up 4TUT+ 4TUT+ at 100 ␮M and UTP as indicated. Reactions were per- p pU pU U Up formed in the presence of 50 nM TbTUT4 for 30 min. Gel Up p UpU 1, the enzyme was preincubated with 32pUpU for 10 UpUp min, and the reaction was started by addition of UTP; gel 2, the enzyme was preincubated with UTP for 10 min, and the reaction was started by addition of 32pUpU; gel 3, UMP and 32pUpU were premixed, and - + - the reaction was started by addition of the enzyme; gel PTU : ,0 1 , 01 , 001 µM ␮ ␣ 32 4, 0.1 M[ - P]UTP and 2 mM UMP were premixed, 23 UpUp UMP iT m :e and the reaction was started by addition of the en- )1( )2( 3( ) 4( ) ,PTU µ :M 1 01 001 1 01 001 1 01 001 zyme. Products were separated on 20% polyacryl- amide/urea gels. (D) Effect of the order of substrate addition on the TbTUT4-catalyzed reaction. Reactions were carried out with 50 nM TbTUT4 in the presence of 0.5 ␮M radiolabeled 6[U] RNA and indicated concentrations of UTP for 1, 5, and 30 min. Products were separated on 15% polyacrylamide/urea gels.

lished data), animal cytoplasmic Gld-2 (13, 18), Saccharomyces Preincubating the enzyme with either substrate before addition of cerevisiae Tr4/5 PAPs (19–21), and the Cid1–13 family of the second reactant significantly decreased the efficiency of nucle- proteins from Schizosaccharomyces pombe (11, 22) (SI Fig. 7). oside transfer. Similar, but less pronounced, effects were observed Our findings suggest that NTP binding by TUTase-like nucle- with a 24-mer 6[U] RNA indicating potential competition of otidyl transferases is relatively promiscuous and that specificity substrates for the binding site. is achieved by additional mechanisms. The crystal structure of TbTUT4 with bound UTP and bound UMP (as the minimal RNA substrate) illustrates triple coplanar RNA Substrate Specificity. RNA substrate specificity differs mark- aromatic stacking of the UTP uracil base sandwiched between the edly among trypanosomal TUTases with RET1 (4, 5), TUT3 (7), phenyl ring of the essential Y189 (8) and the uracil base of the and TUT4 (8) preferentially acting on single-stranded substrates minimal RNA, UMP (Fig. 3A), an arrangement that closely re- and RET2 capable of using double-stranded RNAs (5, 6), (Fig. 2A). sembles an RNA strand in a double helical conformation. The Putative editing intermediates are represented by model RNA direct contacts of the ‘‘terminal’’ RNA residue with the enzyme substrates for a ‘‘precleaved’’ U-insertion assay (Fig. 2A). TbTUT4 consist of three hydrogen bonds with R121, D68, and D136 and is inactive on base-paired RNA substrates but readily extends the hydrophobic interactions of the base with V122 (Fig. 3B). A single-stranded ‘‘5Ј fragment’’ (Fig. 2A Left, lane 2) and 24-mer significant increase in the apparent Km for RNA due to the R121A 6[U] (Fig. 2B Left) substrates. Unexpectedly, RNA with six ad- mutation has been demonstrated previously (8). Taken together, enosines at the 3Ј end, 6[A], displayed much lower efficiency as a these observations suggest that hydrogen bonding of the ribose and primer than 6[U] RNA (Fig. 2B Center). Replacing only the 3Ј uracil base with conserved protein residues, the stacking interaction uridylyl residue with an adenosyl nucleoside had an inhibitory effect with UTP, and coordination of the 3Ј hydroxyl by a second (Fig. 2B Right). We hypothesized that the uracil base at the 3Ј magnesium ion (Mg2ϩ) are all essential for correct positioning of terminus of the RNA primer is involved in specific interactions with the . Because no structure of the homoribonucleotidyl the RNA and, therefore, catalysis. To further analyze the binding transferase with bound RNA is available and our attempts to of the RNA substrate by TbTUT4, an additional UMP residue was cocrystallize TbTUT4 with RNA and a nonhydrolyzable UTP modeled by hand into the ternary TbTUT4:UTP:UMP crystal analog have so far been unsuccessful, we investigated whether structure to form a 2-mer RNA molecule in the active site (Fig. 3C). uridine monophosphate could serve as minimal RNA substrate to The pose of this modeled penultimate RNA residue suggests Ј elucidate RNA positioning adjacent to the UTP binding site. hydrogen bond interactions between the hydroxyl groups of the 5 In contrast to our earlier findings of primer-independent UTP ribose and R126 (K149 in TbRET2), a hydrogen-bonding pattern polymerization by RET1 (23), no such reaction could be detected similar to that observed between N147 and the ribose hydroxyls of for TbTUT4 at various UTP concentrations (data not shown). the bound UTP (8, 14). These interactions based on modeling are Incubation of TbTUT4 with UMP and radiolabeled UTP led to an consistent with the fact that the R126A mutant is virtually inactive accumulation of the pUpU adduct, indicating that UMP indeed because of loss of RNA binding, but not UTP binding (8). Fur- binds at the active site and is capable of assuming the role of an thermore, the hydrogen bond received by O4 of the 5Ј uracil base RNA substrate (Fig. 2C, gel 4). Increasing the size of the RNA suggests that the enzyme prefersaUresidueinthepenultimate substrate to a 2-mer (pUpU) led to an increase in efficiency, but position of the RNA substrate. This could explain in part why only if the reaction was initiated by the addition of the enzyme to TbTUT4 functions more efficiently on a 5[U]A RNA primer than a mixture of the UTP and pUpU substrates (Fig. 2C, gels 1–3). on a 6[A] primer (Fig. 2B).

14636 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704259104 Stagno et al. Downloaded by guest on September 28, 2021 A B C 121R

981Y 621R

PTU V122 Fig. 3. The prereaction complex of UTP and UMP 2ϩ PMU (‘‘RNA’’) in the active site of TbTUT4. Mg ions (black) PMU 631D PMU are labeled Mg1 and Mg2, where Mg1 is the binding M 1g 121R site previously observed in the TbTUT4:UTP structure 2gM M 2g (8). (A) Triphosphate coordination by Mg1 and forma- 1taW 6D 6 PTU 86D tion of a binding site for a second metal ion (Mg2) 86D W 1ta D68 upon UMP binding. (B) Direct protein–UMP hydrogen M 1g 631D bond contacts. (C) Hydrogen bond contacts with UMP 631D Y 981 (‘‘RNA’’) at the terminal (gray) and modeled penulti- mate (green) UMP residues.

RNA Contribution to Selection and Catalysis. In addition and the triphosphate moiety of UTP. Similar observations have to the primary role of RNA as a nucleophile in the transferase been reported for TbRET2 (14). However, upon binding of the reaction, its terminal residue base-stacking interaction with the minimal RNA substrate, UMP, a second Mg2ϩ (Mg2) was ob- bound NTP suggests a role of the RNA in nucleoside incorporation served, which coordinates the two reactants of the transferase selectivity by uridylyl transferases. In the case of purine nucleoside reaction by positioning the 3Ј hydroxyl of the RNA Ϸ4.6 Å away triphosphates, coplanarity between the purine ring and Y189 is from the ␣-phosphorus of the bound UTP (Fig. 5A). In line with a significantly reduced (60° versus 45° in the case of pyrimidines). universal two-metal-ion mechanism for nucleotidyl transfer (24), This, accompanied by a relative translation for purine bases, would Mg2 is expected to facilitate deprotonation of the RNA 3Ј hydroxyl result in virtually no stacking of bound ATP or GTP with the 3Ј base to stimulate the nucleophilic substitution reaction, whereas Mg1 is of the bound RNA substrate (Fig. 4). The resulting reduction in thought to stabilize the leaving group. The impor- binding affinity for the RNA substrate may adversely affect the tance of triple base stacking during catalysis is further confirmed by positioning of its 3Ј hydroxyl group, thereby explaining the reduc- the crystal structure of TbTUT4 with bound UpU, which depicts a tion in the observed catalytic rates for purine incorporation into pseudoproduct state of the transferase reaction between UTP and RNA (Table 1). Discrimination between UTP and CTP, on the UMP (Fig. 5B). The crystal structure reveals that the UpU binds other hand, does not involve a selective RNA binding factor and such that one uracil base takes the place of the UTP uracil and the other takes the place of the RNA (UMP) uracil. The possible bases relies on a highly coordinated water molecule, which accepts a Ј hydrogen bond from the donor-only N3 of the uracil base in the case within range to deprotonate the ribose 3 hydroxyl, a necessary step for catalysis, are D68, D136, and Wat1 (Fig. 5A). However, the of UTP binding (8, 14). direct interactions of D68 and Wat1 with divalent cations would The nucleotidyltransferase reaction involves nucleophilic attack likely make them highly acidic, suggesting that D136 functions as by the 3Ј hydroxyl of the RNA substrate on the ␣-phosphorus of the the catalytic base. This would explain previous observations that, bound NTP, which results in nucleoside incorporation into the although the universally conserved D136 does not contribute RNA and liberation of pyrophosphate. Three conserved catalytic directly to NTP or Mg2ϩ binding, its mutation to alanine renders the aspartates coordinate the divalent metal ions essential for catalysis. RET1 and TUT4 TUTases inactive (8, 23). The crystal structure of TbTUT4 with bound UTP (8) revealed a 2ϩ Upon UTP binding, TbTUT4 undergoes significant conforma- single Mg (Mg1) coordinated by two aspartates (D66 and D68) tional changes that bring the N-terminal and C-terminal domains closer together into a more compact structure (8). On the other hand, the TbTUT4 protein conformations in the UTP-, UTP:UMP-, and UpU-bound structures are virtually identical, suggesting that neither RNA binding to the TbTUT4:UTP binary complex nor catalysis is accompanied by major conformational BIOCHEMISTRY

Fig. 4. Triple-stacking interaction is required for productive RNA binding. Stereo views of the superposition of TbTUT4:ATP and TbTUT4:UTP:UMP, respec- tively, illustrate the various degrees of stacking of the aromatic rings of Y189, the Fig. 5. Structures of the active sites of the pre- and postreaction complexes. (A) NTP base, and UMP (RNA) for purine NTPs (adenine, shown in green) versus Reactant state with TbTUT4:UTP:UMP. Mg2 (black sphere) positions the 3Ј hy- pyrimidine NTPs (uracil, shown in yellow). Upon superposition, there is virtually droxyl of UMP (terminal RNA residue) for in-line nucleophilic attack on the no base stacking observed between the pyrimidine ring of UMP and the purine ␣-phosphorus atom of the bound UTP at a distance of 4.6 Å. (B) state with ring of ATP. TbTUT4:UpU.

Stagno et al. PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 ͉ 14637 Downloaded by guest on September 28, 2021 A B C for RNA binding (8) and the enzyme’s preference for a terminal UM :P TbT TU 4 f a ov der :PMA Tb ER T2 af v dero uridylyl residue. Superposition of this structure with the crystal bT 2TER structure of TbRET2:UTP (14) (PDB ID code 2B51) exposes a 6 U[ ] [6 ]A A]U[5 E3 424E/00 4E/003E 24 potential clash at the O4 position with E424 (RET2) (Fig. 6B), a key

R 1R/121 44 441R/121R residue involved in UTP binding (14). However, modeling an AMP in the same relative position as the bound UMP (as observed in the R1 14 V/ 271 R141 V/ 2 17 structure of TbTUT4:UTP:UMP), assuming that the terminal RNA residue is fixed because of maximized stacking with the uracil base and the constraints for the position of the 3Ј hydroxyl, suggests 38C/25F 38C/25F an interaction between E424 (TbRET2) and the amino group of the c PTU c PTU c PTU adenine base (Fig. 6C). Furthermore, clashes are evident between

PMU PMA active site residues of TbTUT4 and the modeled AMP, suggesting that binding of RNA substrates ending in A is less energetically Fig. 6. RNA specificity of trypanosomal TUTases. Potential stabilizing inter- favorable for this enzyme. Therefore, in addition to coordinating actions are shown as blue dotted lines, and destabilizing electrostatic repul- the UTP uracil base, two additional roles for E424 in RET2 are sion and steric hindrance are shown as solid and dotted black arrows, respec- likely: (i) contributing to RNA substrate specificity by energetically tively. (A) TbRET2 is highly selective for the terminal nucleoside in a single- favoring an adenosine as the terminal RNA residue and (ii) limiting stranded RNA substrate. (B) Superposition of the TbTUT4:UTP:UMP and nucleoside incorporation by RET2 into single-stranded RNA to TbRET2:UTP (PDB ID code 2B51) crystal structures. Key residues of TbTUT4 one U residue. (purple) and TbRET2 (cyan) are indicated. (C) Same as in B but with UMP replaced by a modeled AMP molecule in the same position. Discussion The fundamental question of how specific nucleoside triphosphates changes of the protein. With the UpU adduct, or any ϩ1 RNA, are recognized by template-independent nucleotidyl transferases occupying the active center as described above (Fig. 5B), a subse- has been investigated for several types of enzymes. Structural quent round of U-incorporation would require a protein confor- analysis of the apo and substrate-bound CCA-adding enzymes mational change to translocate the new 3Ј OH of the RNA into revealed that both class I and II proteins use hydrogen bonding position for nucleophilic attack. An alternative scenario is the complementarity between the Watson–Crick edge of the incoming dissociation of the entire RNA molecule or its 3Ј end from the NTP and conserved amino acid residues. In the case of class I protein and reassociation, concurrent with or subsequent to UTP CCA-adding enzyme from Archaeoglobus fulgidus (AfCCA), base binding. Also possible is active displacement of the RNA back to the recognition is also aided by stacking of the incoming NTP on the primer (UMP) binding site due to competition with the incoming growing RNA primer, as well as contacts with RNA phosphate UTP molecule, which is likely to have a higher affinity for this site groups (26). The eukaryotic nuclear PAP, which is structurally as a result of extensive triphosphate contacts. Preincubation of the homologous to class I archaeal CCA-adding enzymes, has been enzyme with either UTP or 32pUpU before addition of the second cocrystallized with ATP analogs under a variety of conditions substrate has a strong inhibitory effect (Fig. 2C, gels 1 and 2) as (27–29) and subjected to extensive mutational analysis (29, 30). However, a coherent mechanism of ATP selection remains to be compared with reactions initiated by the addition of enzyme to a established. Furthermore, a highly organized ATP binding site has mixture of both substrates (Fig. 2C, gel 3). Longer RNA substrates, been reported for vaccinia virus PAP (31). Terminal uridylyltrans- which are more physiologically relevant, may induce conforma- ferases belong to the same, apparently monophyletic group as tional changes upon catalysis that facilitate reformation of the apo nuclear PAPs and archaeal CCA-adding enzymes (15) but are most state. However, the ‘‘order of addition’’ effects observed with a closely related to divergent, ‘‘noncanonical’’ PAPs, such as animal longer RNA substrate resemble those observed with pUpU (Fig. 2 Ј ϩ Gld-2 type cytoplasmic PAP, Trf4/5 nuclear surveillance PAPs in C and D), supporting a model in which the 3 end of 1 RNA S. cerevisiae, and the Cid1-like protein family in S. pombe (1). The partially dissociates from the active site and reassociates in concert Ј high-affinity NTP binding site of trypanosomal TbTUT4 is capable with UTP binding. The base-specific contacts of the 3 and pen- of selective UTP binding versus ATP binding at submicromolar ultimate nucleosides (Fig. 5) are most likely crucial for such NTP concentrations (8). Because of the conservation of protein– reassociation. Indeed, for a relatively small protein, such as UTP contacts between TbTUT4 and TbRET2, this conclusion is TbTUT4, which does appear to possess an extended RNA binding likely to be applicable to other TUTases. surface adjacent to the active site, processivity may be achieved by In the present work we demonstrate that, at higher, more Ј relatively few contacts with 3 terminal RNA residues. physiologically relevant concentrations, all four NTPs can occupy the active site of TbTUT4 with nearly perfect superposition of the Structural Bases of Terminal Nucleoside Selectivity. Although the phosphate groups. The implication of this finding is twofold: (i) physiological RNA substrates for TbTUT4 are currently unknown, other mechanisms, in addition to NTP binding affinity, are required it is clear that this enzyme is selective for RNAs that contain Us at to discriminate non-UTP substrates by TUTases, and (ii) TUTase- the 3Ј terminal and penultimate positions (Fig. 1). Conversely, like bidomain core modules are quite promiscuous in NTP binding; recombinant TbRET2 is inactive with an RNA primer bearing six a minimal number of mutations is likely to suffice to convert this Us at the 3Ј end, whereas an RNA with a terminal adenosine, module into an ATP-specific [noncanonical poly(A)] polymerase or 5[U]A, is extended as efficiently as a substrate bearing six ad- vice versa. The necessity for continuous stacking interactions enosines, 6[A] (Fig. 6A). RNAs that have been extended by a single between a conserved tyrosine side chain, the bound NTP, and the uridylyl residue are no longer active in the TbRET2-catalyzed terminal nucleoside base of the RNA primer poses a constraint on reaction. Thus, for TbRET2, as well as for TbTUT4, the terminal the positioning of the NTP base. This may reflect principal differ- nucleoside provides a significant contribution to productive RNA ences in NTP selection between TUTases and CCA-adding en- binding due to base-specific contacts. Such specificity likely reflects zymes. In AfCCA, tRNA forms part of the nucleotide binding site; the purine-rich nature of preedited mitochondrial mRNAs, which the apo enzyme is unable to discriminate correct substrates (CTP are extended by RET2 upon endonucleolytic cleavage (for a review and ATP) from incorrect ones (UTP and GTP). In TUTases, the see ref. 25). Structural analysis of the TbTUT4:UTP:UMP ternary enzyme has an intrinsic selectivity for UTP in the absence of RNA, complex reveals an interaction between R121 and O4 of the UMP and base-specific contacts are essential for UTP binding (8), uracil base, thereby explaining both the essential role of this residue suggesting that protein–UTP affinity is required but not sufficient.

14638 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704259104 Stagno et al. Downloaded by guest on September 28, 2021 In the case of ATP or GTP, steric constraints prevent the purine a suboptimal substrate for RET2. This self-limiting mechanism may base from participating in these stacking interactions, which is have a role in the overall fidelity of U-insertion RNA editing by thought to interfere with productive RNA binding. The significant minimizing the number of nonguided U-insertions. drop in catalytic rates for purine NTPs, which is consistent with the loss of optimal substrate orientation, points to a kinetic component Materials and Methods in the selection process. The structure of ATP-bound TbTUT4 Uridylyltransferase Activity Assays. Reactions of purified recombi- suggests that, in TUTase-like PAPs, the aspartate at position 297, nant proteins with synthetic RNA substrates were performed an essential residue for uracil-specific hydrogen bonding of Tb- as described previously for TbRET2 (5) and TbTUT4 (8). The TUT4 (8), would play no role in , whereas S148, SigmaPlot software package was used for calcu- Y189, and N147 remain important for catalysis (Fig. 1). lations of Km, Vmax, and standard deviations. Reactions with UMP Selectivity of TbTUT4 and TbRET2 toward the terminal RNA and UpU were separated on a 20% acrylamide gel and exposed base is likely to be dictated by stabilizing effects of direct hydrogen immediately to a phosphor storage screen for 30 min. UMP and bonding with conserved residues, as well as destabilizing electro- UpU were obtained from Sigma (St. Louis, MO). Single-stranded static repulsion and steric hindrance effects (Fig. 6). Processive RNA substrates were 6[U] (GCUAUGUCUGUCAACU- TUTases, such as RET1 (23), TUT3 (7), and TUT4 (8), favor UGUUUUUU), 6[A], (GCUAUGUCUGUCAACUUGAA- oligouridylyl RNA primers while displaying a lesser specificity AAAA), and 5[U]A (GCUAUGUCUGUCAACUUGUUU- toward UTP than RET2 (5, 6). This may be explained by our UUA). Double-stranded RNAs were used as described in ref. 4. modeling studies (Fig. 6): in TbTUT4, the arginine at position 141, which is conserved among processive TUTases (1), is locked in a Crystallization, Data Reduction, and Refinement. Purified TbTUT4 salt bridge with E300, thus precluding participation of the gluta- was concentrated to 5 mg/ml in 10 mM Hepes buffer (pH 7.6), 70 mate in the coordination of the crucial water molecule that makes mM KCl, and 0.5 mM DTT and crystallized in the presence of 4 ␮ a uracil base-specific contact (8, 14). Furthermore, R141 likely mM MgCl2 and 25 M of the respective NTP. Crystallization disfavors adenosine binding because of a clash with the exocyclic conditions were 100 mM sodium cacodylate (pH 6.5), 200 mM amino group. In TbRET2, the equivalent position (271) is occupied calcium acetate, and 18% PEG-8000 (Crystal Screen solution 46; by a valine instead of an arginine, providing a more spacious binding Hampton Research, Aliso Viejo, CA) and were carried out at 4°C site and resulting in a change of position of the side chain of the using the vapor diffusion method. A TbTUT4-UTP cocrystal was aforementioned glutamate, E424 (E300 in TbTUT4). E424 in soaked in mother liquor containing 10 mM UMP and another in 10 TbRET2 thus plays a role in favoring adenosine over uridine as the mM UpU for 30 min at 4°C, which were used to derive the terminal RNA nucleoside and allows for exquisite UTP specificity. structures of the TbTUT4:UTP:UMP ternary complex and TbTUT4:UpU, respectively. All crystals were flash-cooled with Arginine-121 is essential for TbTUT4 activity (8) and acts as a liquid N in mother liquor supplemented with 25% glycerol as a positive determinant for terminal RNA uracil binding while dis- 2 cryoprotectant. All x-ray data were collected at the Stanford Linear criminating against adenosine. The phenylalanine in position 52 of Accelerator Center and processed and scaled by using d*TREK TbTUT4 is conserved for RET1 and TUT3 among all kinetoplas- (32). The structures were solved by refining the original tids but is replaced with a smaller cysteine in RET2, perhaps further TbTUT4:UTP structure (PDB ID code 2IKF) against the structure enhancing binding of a purine base. Our model provides a rationale factors obtained from each data set while taking care to conserve as to why RET2, acting on a single-stranded RNA substrate, adds the test set. Model building and refinement were carried out by only a single uridylyl residue. However, in vivo, some substrates of using the programs COOT (33) and REFMAC5 (34), respectively. RET2 are likely to be two double-stranded RNAs linked by purine Ј nucleotides (Fig. 2A). Here the extended 5 fragment reanneals We thank James Weng for excellent technical assistance and members with guide RNA, thus restoring an optimal RNA substrate for each of the R.A. laboratory and the H.L. laboratory for discussions. This work round of addition. Consequently, a mismatched addition product was supported by National Institutes of Health Grants AI064653 (to becomes a single-stranded RNA with U at the 3Ј end and therefore R.A.) and GM56445 (to H.L.) and a Chancellor’s Fellowship (to H.L.).

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