Crucial Roles of Two Hydrated Mg2+ Ions in Reaction Catalysis of the Pistol Ribozyme

Abstract Pistol ribozymes constitute a new class of small self‐cleaving RNAs. Crystal structures have been solved, providing three‐dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage, corresponding to the pre‐catalytic state, a vanadate mimic of the transition state, and the product. The results led to the proposed underlying chemical mechanism. Importantly, a hydrated Mg2+ ion remains innersphere‐coordinated to N7 of G33 in all three states, and is consistent with its likely role as acid in general acid base catalysis (δ and β catalysis). Strikingly, the new structures shed light on a second hydrated Mg2+ ion that approaches the scissile phosphate from its binding site in the pre‐cleavage state to reach out for water‐mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg2+ ion appears to be the stabilization of product conformation. This study delivers a mechanistic understanding of ribozyme‐catalyzed backbone cleavage.

Thea ssessment of ribozyme X-ray structures is demanding because these molecules require structural flexibility at the cleavage site and active-site pocket for the spatio-temporal correlation to enable the chemical reaction. Therefore,c areful structure-function analysis in solution by targeted mutagenesis is necessary.C omplementarily,apotent approach to advance our understanding of ribozyme catalysis is the structure elucidation of transition-state (TS) analogues.T his approach is,h owever,achallenging experimental task.
Suitable mimics for the pentavalent TS of ap hosphorane are rare and those of vanadate analogues have to date been solved for hairpin and hammerhead ribozymes only. [28][29][30] In the present study we set out to obtain conformational snapshots along the reaction coordinate of the pistol ribozyme phosphordiester cleavage ( Figure 1). We succeeded in solving the X-ray structures of both its TS analogue vanadate (at 2.8 resolution) and the ternary 2',3' cyclophosphate product complex (at 2.65 resolution;F igure 2; see Fig- ure S1 in the Suppporting Information for stereoviews). Together with our previously obtained structure of the precleavage conformation of the pistol ribozyme at 2.7 resolution [24] (Figures 1c and 2a), we achieved an architectonic framework for the cleavage reaction, allowing profound proposal for the underlying chemical mechanism. In short, ad ivalent ion (Mg 2+ ,M n 2+ )s trictly remains innerspherecoordinated to N7 of G33 in all three states (pre-cleavage, [24,31] TS analogue,and post-cleavage) and allows simultaneous water-mediated interaction with the pro-R nonbridging oxygen atom of the scissile phosphate.S trikingly,asecond hydrated Mg 2+ ion moves from its binding pocket in the precleavage state towards the scissile phosphate in the TS and becomes coordinated between the pro-S oxygen atom and the N7 of A38 in the product. Our findings suggest the potential participation of ahydrated Mg 2+ as ageneral acid for proton transfer to the 5'Ol eaving group (d catalysis) in pistol ribozyme cleavage,a nd also suggest an additional role for as econd Mg 2+ in the conformational stabilization of the product.
Most X-ray structures of small self-cleaving ribozymes refer to the precatalytic fold, with the nucleoside 5' of the scissile phosphate substituted by the corresponding 2'-deoxynucleoside to prevent cleavage.T his arrangement was also the case for our recently solved structure of the pistol ribozyme ( Figure 2a). [24] Modelling of ah ydroxy group onto the 2'-deoxyribose revealed an "in-line" orientation of this 2'O, ready for attack at the phosphorus atom of the "to-becleaved" P À O5' bond, which is in accordance with mechanistic requirements (S N 2-like). Furthermore,evaluation of the structure by atomic mutagenesis experiments pointed to the crucial role of ad ivalent cation that was innerspherecoordinated to N7 of an active site guanine (G33). [31] The assignment of am etal ion coordinated to N7 of purine nucleobases can be ambiguous at aresolution of 2.7 , [32] and hence,efforts were made to verify the metal ion coordination by Mn 2+ soaking.T he obtained anomalous electron-density map and the coordination distance of 2.1 were supportive for ad ivalent metal binding site. [24] Most importantly,o ur finding that deletion of the coordination site (mutation of G33 to 7-deazaG33) rendered the ribozyme almost completely inactive,even in the presence of high concentrations of Mg 2+ ions,s uggested an important role of Mg 2+ in catalysis. [31] We note here that the distance of the N7-coordinated Mg 2+ to the pro-R oxygen atom of the scissile phosphate was 4.4 and is consistent with aw ater-mediated hydrogen-bond interaction as indicated in Figures 2a,b.
To increase our understanding of the pistol ribozymes catalytic mechanism, we have now determined its structure when complexed with aT Sm imic at 2.8 resolution. To obtain the crystal structure of the TS analogue,wemixed the three RNAstrands shown in Figure S2. Theresulting complex lacks the scissile phosphate but retains the 2'-, 3'-, and 5'hydroxy groups at the cleavage site.T his complex was cocrystallized with NH 4 VO 3 .D etails are provided in the Supporting Information (see Tables S1 and S2). Theelectron density in the active site can be assigned to vanadate based on its size,shape,and anomalous scattering ( Figure 3). Distances between the 2'-, 3'-, and 5'-oxygen atoms and the vanadium atom are consistent with direct coordination between the oxygen atoms and the vanadium atom.
Thes tructure of the TS analogue reveals fine but significant rearrangements relative to the pre-cleavage structure ( Figure 2c,d). Most importantly,w hile the key players (ribose-32, G33, G40, G53, U54) in the active site only minimally alter their positions,t he conformation of the backbone of the cleavage site is changed. Then onbridging oxygen centers of the scissile phosphate are rotated, bringing both the pro-S oxygen and pro-R oxygen centers much closer to the Hoogsteen face of G33 (shifting from 6.2 to 3.6 and from 4.4 to 3.3 towards N7-G33, respectively;Figure 2a,c). As ar esult, the divalent metal ion (whose innersphere coordination to N7-G33 was verified by the anomalous signal in the structure of Mn 2+ soaked crystals; Figure 3) potentially interacts through water-mediated hydrogen bonding with the nonbridging oxygen atoms of the scissile phosphate.T his ion is also at a4 distance to the 5'Oleaving group of U54 such that it could position aw ater molecule to serve as ag eneral acid, in the cleavage reaction, for proton transfer to stabilize the leaving 5'O( Figure 2c,d).
Furthermore,our TS analogue structure strongly suggests the bidentate interaction of G40 with the phosphorane TS during cleavage.I nt he vanadate complex both, N1 of G40 and N2 of G40 are within hydrogen-bonding distance to the 2'O( 2.5 )a nd the pro-R oxygen atom (3.4 ), respectively (Figure 2c,d). Ad irect role of G40 as abase in general acidbase catalysis is widely accepted according to functional mutagenesis assays, [24,31,33] and it is now complemented and consistent with the structural framework provided here.W e also note that the cleavage rate differences observed for phosphorothioate substrates are consistent with our structures. [33,34] Thesignificantly reduced rate measured for the R P diastereomer is in agreement with adirect recognition of the phosphorothioate moiety by G40. Furthermore,b ecause the activity was not restored by addition of Mn 2+ ions, [35,36] watermediated (outersphere) rather than innersphere coordination between the phosphorothioate and the metal ion is likely and the M 2+ -'proR-O' distance of 3.3 in our TS structure supports this notion further. In contrast to the R P diastereomer, cleavage of the S P diastereomer was only minimally diminished and this finding is also plausible because the pro-S oxygen atom has no direct interactions in our structures of the TS analogue and the pre-cleavage state.
Thet hird structural snapshot on the pistol ribozyme reaction coordinate that we have solved represents the postcleavage state (Figure 2e,f). In this product structure,G 53 and U54 are slightly more distant to each other compared to the TS analogue.M oreover,t he ribose moiety of G53 finds itself in ad istinctly different orientation, with its 2',3'-cyclic phosphate rotated relative to the vanadate moiety in the TS mimic. As ar esult, the cyclic phosphate is recognized by a2 .6 hydrogen bond between G40-N1 and its pro-S nonbridging oxygen atom, and a3 .0 hydrogen bond between the free 5'OH and its nonbridging pro-R oxygen atom. Notably,the divalent metal ion remains coordinated to the N7 of G33 (2.3 )a nd its 2.8 distance to the pro-R oxygen of the cyclic phosphate offers the possibility for aw ater-mediated interaction.
Importantly,t he structure of the pistol product complex clearly shows as econd metal ion that was located at a4 .0 distance to the pro-S oxygen atom of the cyclic phosphate, and therefore also offers the possibility for outershell, watermediated hydrogen bonding.Asecond divalent metal ion was also present in the TS analogue and the pre-cleavage structures,h owever, with greater distances of 4.8 and 6.3 ,r espectively.C onsequently,t his ion and the scissile phosphate approached each other along the reaction coordinate as illustrated in the superposition of the three structures in Figure 4a nd Figure S3.
According to the new product and TS analogue structures, and the formerly solved precatalytic pistol ribozyme struc-ture,p ossible (outershell) coordination sites for this second hydrated metal ion are A38 (N7 and phosphate;h ighly conserved as purine) and A39 (N7;n ucleotide identity not conserved;F igures 2a,c,e). We therefore analyzed whether deletion of the N7 coordination sites,byreplacement with 7deazaadenosine (c 7 A; atomic mutagenesis), has an impact on ribozyme activity ( Figure 5). By applying ap reviously established fluorescence spectroscopic assay (based on A57Ap substrate labeling), [24,31] cleavage kinetics were determined. Thec leavage rate was comparable to the wildtype for the A38c 7 Amutant, 1.5-fold reduced for the A39c 7 Amutant, and 8-fold reduced for the A38c 7 A-A39c 7 Ad ouble mutant (Figures 5c-f and Table 1).
Alternative assays using HPLC analysis independently confirmed this behavior (see Figures S4 and S5). Clearly,the effect of deletions of these potential N7 coordination sites is minor when compared to the more than 1000-fold rate reduction observed for the G33c 7 Gm utant (Table 1). Furthermore,t he fact that the coordination of the ion to the scissile phosphate is solely observed in the product state suggests am ore subtle role in phosphodiester cleavage.W e speculate that ar eason for this interaction in the product

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Research Articles could be stabilization of ac yclophosphate conformation that hinders the reversible reaction (ligation).
By further revisiting the pre-cleavage structure with respect to the exact location of this Mg 2+ ion, we noticed that the O6 of G40 is also at ad istance that would allow outershell interactions,a nd hence,m ay contribute to activation of G40 (the general base). Since this hypothesis has not been experimentally challenged in former studies, [31,33] we set out to synthesize aG 40Ap mutant which lacks the O6, but otherwise provides the imine N1 and amine N2 (as encountered in the enol form of G; Figure 6a). Qualitative HPLC cleavage assays indicated that the G40Ap mutant cleaved as fast as the wildtype ribozyme (Figure 6b,c). Although the G40Ap mutant did not allow application of our former realtime fluorescence assay for precise rate determination, because of interference with A57Ap,w ea nalyzed the fluorescence response of the G40Ap mutant itself.W e observed ad ecay in response to Mg 2+ addition, with an apparent rate of 4.2 min À1 ,w hich was comparable to that of the wildtype ribozyme ( Figure 6e and Table 1). In contrast to G40Ap,the (O6 containing and N2 lacking) G40I mutant was significantly slower in cleaving (Figure 6d), with ar ate of 0.018 min À1 only (ssee Figure S6 and Table 1). This 220-fold rate reduction provides evidence for ad ominant role of the G40-N2 functional group in TS recognition (as discussed above).
Another nucleotide,w hose role in pistol ribozyme cleavage has been intensively debated, is G42. [18,31,33] In the precatalytic structure,t his guanine mediates the formation of ac left (to accommodate the cleavage site), characterized by the hydrogen bond between the G42 N2 and the 2'Oo f ribose-32 (2.7 ), and by locating its O6 nearly equidistant to the G40 N2 (2.8 )a nd dG53 N2 (2.7 ;s ee Figure S7a). In the TS analogue,G 42 retains the interaction with ribose-32 (2.4 )b ut simultaneously releases G53 and G40 (see Figure S7b). In the product structure,G 42 is redirected towards G40 (2.8 distance between G42-O6 and G40-N2). Simultaneously,t he cyclophosphate terminus of G53 slides away,w ith its N2 in over 4.4 distance to O6-G42 (see Figure S7c). We underline that the strong link between the 2'-OH of ribose-32 and the exocyclic (Watson-Crick) NH 2 of G42 is retained in all three structures (see Figure S7). The crucial role of the ribose-32 2'OH is consistent with signifi-cantly reduced activities when this group was replaced by either H, OCH 3 ,orN H 2 . [24,31,33,37] Our new structures of TS analogue and product significantly improve the mechanistic understanding of pistol ribozyme phosphodiester cleavage,f or which we postulate the following scenario:Inthe precursor,adivalent ion (Mg 2+ ) becomes innersphere-coordinated to N7 of G33 and is additionally held in place by first shell water-mediated hydrogen-bond interactions,o ne to the 2'Oo fr ibose-32, and the other one to the pro-R oxygen of the scissile phosphate (Figure 2b). At the same time,G 40 assists in proton transfer from the G53 2'O, which attacks the scissile phosphorus atom in-line to the PÀO5' bond. In the TS,t he nucleobase of G40 stabilizes the phosphorane in abidendate   manner (via N1···2'Oand N2···pro-R O), and simultaneously, the proximity of the divalent ion that remains innerspherecoordinated to N7 of G33 assists in lowering the energy barrier by electrostatic interactions and by outershell coordination to the pro-R nonbridging oxygen atom of the scissile phosphate (Figure 2d). This process,inturn, places one of its first shell water molecules into appropriate distance for proton transfer (general acid) to the 5'Ol eaving group (Figure 2d). Ther esulting cyclic phosphate product is embedded in anarrowed hydrogen-bond network involving two divalent ions (Mg 2+ )i no utersphere coordination to pro-R and pro-S oxygen centers,r espectively (Figure 2f). This arrangement may contribute to the stabilization of ac yclophosphate conformation that prevents ligation of the cleaved fragments (reversible reaction).
Only two other ribozymes have been structurally characterized by TS analogues so far. Fort he hairpin ribozyme, av anadium oxide mimic was utilized for the first time. [28] Direct interactions with nucleobase functional groups,w hich appeared to stabilize the electronic structure and geometry of the TS,h ad been revealed, [28] as well as potentially involved water molecules. [29] Comparable to pistol ribozyme,t he superposition of pre-cleavage,T Sa nalogue,a nd product structures of hairpin ribozyme showed that the active site was essentially rigid, with motion confined to the scissile phosphate and the ribose pucker of the nucleotide upstream. Distinct from the pistol ribozyme,h owever, was that no divalent metal ion was present in the hairpin ribozyme active site and only nucleobases contributed to recognition of the cleavage site.
More recently,t he crystal structure of ah ammerhead ribozyme (HHRz) TS analogue was reported. [30] This vanadate complex revealed significant rearrangements compared to the previously determined pre-cleavage HHRz structures. Thea ctive site contracted, bringing ag uanine (G10.1) closer to the cleavage site ( Figure 7). This guanine resembles G33 in the pistol ribozyme,a nd similarly,i tc oordinated ad ivalent ion through N7 and ab ackbone phosphate (A9). This ion came closer to the scissile phosphate (3.9 ). Although the distance is farther compared to the situation in pistol, the HHRz vanadate structure also suggested acontribution to TS stabilization through water-mediated interaction with the scissile phosphate.Asecond divalent ion was observed to be innersphere coordinated to O6 of ag uanine,w hich is considered the general base in the hammerhead ribozyme active site (G12). This metal ion likely helps tune the pK a value of G12 to be appropriate for activation of the C17 2'On ucleophile.W ea lso note that deletion of O6 in aG 12Ap mutant of the hammerhead ribozyme caused ad ramatic rate reduction (in the order of 10 3 ), [38] which reflects the significance of innersphere coordination of the metal ion to the G12 O6. In contrast, the second Mg 2+ in the pistol ribozyme active site plays am inor role with respect to rate enhancement, but instead assists in conformational stabilization of the cyclic phosphate in the product complex.
In summary,o ur vanadate and product structures of the pistol ribozyme provide an unprecedented architectonic framework that sheds new light on this ribozymesm echanism. Ad ivalent hydrated metal ion that is innersphere coordinated to N7 of G33 in all three states (pre-cleavage, transition state mimic,p ost-cleavage) consolidates its major role in d and b catalysis,and provides arationale for the more than 1000-fold loss in activity if its coordination to G33 is impaired. Thecleavage is further supported by asecond Mg 2+ which stabilizes the 2',3'-cyclic phosphate in the product complex. Theh ere provided structural framework may also stimulate further computational work on the pistol ribozyme mechanism. [39] Accession codes. Protein Data Bank (PDB): atomic coordinates and structure factors have been deposited under the following accession codes:6 UEY for pistol ribozyme TS analog vanadate,6 UFJ for its 2',3' cyclophosphate product complex, 6UF1 for TS analog vanadate crystals soaked in Mn 2+ ,and 6UFK for the 2',3' cyclophosphate product crystals soaked in Mn 2+ .