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May 9, 2017 - neutrality, however, and thus nonideal for general acid−base catalysis. ... this method on data from the hepatitis delta virus ribozym...
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Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17 Erica A. Frankel,†,‡ Christopher A. Strulson,†,‡,∥ Christine D. Keating,† and Philip C. Bevilacqua*,†,‡,§ †

Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, United States Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States § Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡

S Supporting Information *

ABSTRACT: General acid−base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid−base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the −1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate−pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate−pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the −1 nucleotide, C17, is increased a remarkable 3.5−5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation−π interactions. Rate−pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

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raphy, and theory have deepened our understanding of small RNA enzymes. In the HDV ribozyme, our laboratory helped establish that the pKa of C75, which is poised to serve as the general acid for self-cleavage, is shifted from 4.2 toward neutrality by ∼4 units6 using favorable electrostatics to drive pKa shifting.18 Other studies from our laboratory demonstrated that hydrogen bonding can aid pKa shifting, in which a base quartet drives pKa shifting of a C in beet western yellows virus (BWYV) RNA to 8.15.19 In addition, studies by Wöhnert and colleagues show that cytosine can functionally and structurally replace uridine in the U-turn motifs found in the anticodons and T-loops of tRNAs by shifting its pKa upward past 8.2 to promote hydrogen bonding with the backbone phosphate.20 Also, studies by Al-Hashimi and co-workers uncovered pKa shifts of C to ≥7.2 in transient GC Hoogsteen base pairs in dsDNA.21 In the hairpin ribozyme, A10, A22, and A38 were shown to have pKas shifted from ∼3.5 to 6.6, 7.2, and 5.9,

atalytic RNAs, or ribozymes, perform diverse roles in biology. The first ribozymes to be discovered were large RNAs (≳150 nucleotides), the group I intron and RNase P, which self-splice and process the 5′-end of tRNA. 1,2 Mechanisms of these and other large ribozymes have been extensively studied.3−5 These ribozymes use an exogenous nucleotide or distal hydroxyl as the nucleophile, leave 2′,3′hydroxyl and 5′-phosphate termini in their products, and are largely metalloenzymes. Small nucleolytic RNA enzymes (≲150 nucleotides), on the other hand, catalyze site-specific cleavage of the phosphate backbone through in-line attack of an adjacent 2′-hydroxyl as the nucleophile, leave 2′,3′-cyclic phosphate and 5′-hydroxyl termini in their products, and employ both metal ion and nucleobase catalysis. For the activation of the 2′-OH, a general base can accept the proton of the 2′-OH and likewise a general acid donates a proton to the 5′-leaving group. Analysis of rate−pH profiles has provided insight into the mechanism of a number of small ribozymes. Among these are the hepatitis delta virus (HDV), hammerhead, Varkud Satellite (VS), hairpin, glmS, twister, twister sister, hatchet, and pistol ribozymes.6−9 Companion studies of mutagenesis, crystallog© XXXX American Chemical Society

Received: February 25, 2017 Revised: April 19, 2017

A

DOI: 10.1021/acs.biochem.7b00174 Biochemistry XXXX, XXX, XXX−XXX

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Figure 1. Secondary structures for the two ribozymes studied herein and hammerhead ribozyme crystal structures. (A) Hammerhead 16 (HH16) minimal ribozyme45 and (B) hepatitis delta virus (HDV).6,33 The HH16 is a two-piece system with separate enzyme and substrate strands, and the HDV ribozyme is a one-piece system. In the HH16 ribozyme, the putative general base, G12, is colored blue and a proposed general acid G8 is colored red. Other bases important in HH16 catalysis are U6 (green) and C17 (pink). In the HDV ribozyme, the general acid C75 is also colored red; the general base, [Mg(H2O)5(OH−)]+ for HDV, is not depicted. Arrows indicate the site of cleavage in both ribozymes. These colors are used throughout the figures and crystal structures. Crystal structures of the ground state hammerhead ribozyme at (C) pH 5 and (D) pH 8. (E) Crystal structure of the hammerhead ribozyme at pH 8.2 bound to a vanadate transition state mimic.34 Distances from M2 to N7 of G12 are 2.4 and 4.3 Å in the pH 8 and 8.2 structures, respectively (not shown for the sake of clarity).

respectively, with A38 positioned to serve as the general acid.12 In the HDV, hairpin, and glmS ribozymes, the pKa values obtained from rate−pH profiles were verified by direct monitoring of the population of protonated cytosine using Raman spectroscopy on crystals, via Raman crystallography.11,22−24 The study presented here focuses on the hammerhead ribozyme (Figure 1A), which was the first small ribozyme to be discovered.25 Identified in the tobacco ringspot virus, the hammerhead ribozyme is often involved in viral rolling-circle replication.26 It is generally found as a one-piece ribozyme in nature, although exceptions have been noted,27 and it occurs both in prokaryotes, such as various forms of bacteria,29,28 and in eukaryotes, such as crickets and humans.27,30 It has been studied in both minimal and native formats, where the latter contain long-range tertiary interactions that stabilize the fold.31 In the lab, the ribozyme can be readily engineered into separate enzyme and substrate strands, where a shorter, chemically synthesizable substrate strand facilitates chemical study. Sequence requirements have revealed 15 conserved bases in the hammerhead ribozyme.32 The majority of the small nucleolytic ribozymes have a specific guanine poised to participate in proton transfer with the nucleophilic 2′-OH.5,10,35−36 However, the unshifted pKa value for guanine is far above neutrality at ∼9.4. It thus becomes important to elucidate driving forces for activation of the 2′-OH nucleophile and whether it would favor catalysis to have the guanine shift its pKa toward or even away from neutrality. Recently, a series of hammerhead ribozyme crystal

structures were determined by Mir, Golden, and co-workers, and these provide insightful views of the ribozyme under different conditions.34,38 Structures of crystals grown as a function of pH were determined and include a pH 5.0 structure, a pH 8.0 structure, and a pH 8.2 transition state analogue structure (Figure 1C−E). Under all pH conditions, a divalent metal ion (M1) is not far from the 5′-O leaving group and may move toward it during the course of the reaction to interact with it directly or indirectly.39,40 A second divalent metal ion (M2) appears exclusively in the two high-pH structures. M2 as a crystallographic Mn2+ ion is 3.3 Å from O6 and 2.4 Å from N7 of G12 at pH 8 and 2.6 Å from O6 and 4.3 Å from N7 at pH 8.2 and thus has been suggested to stabilize the deprotonated form of G12.9,38,42−41 In an effort to elucidate interactions between metal ions and catalytic nucleobases, as well as identify catalytic roles of various species, we measured and globally fit rate−pH profiles for the hammerhead ribozyme over a wide range of Mg2+ concentrations. We report cooperative interactions among G12, C17, and Mg2+ ions. Our finding supports the notion that C17 is cationic at neutral pH and performs a redundant role with M2 in influencing the deprotonation of G12. Moreover, under biological conditions, the major reactive channel appears to involve a protonated C17.

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EXPERIMENTAL PROCEDURES Materials. Hammerhead substrate strand RNA oligonucleotides were obtained from Integrated DNA Technologies (IDT, Coraville, IA) and purified as described below. Transcription B

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as averages, with the error being the standard deviation. All reactions were conducted under single-turnover conditions. A saturating enzyme concentration of 0.1 μM was used45 with trace radiolabeled substrate (picomolar). Reactions were typically monitored for 2−4 h. Fitting of Kinetic Data for the HH16 Ribozyme. Kinetic data were collected as a function of pH, parametric in Mg2+ concentration. Data were fit to one of three kinetic models, as described in Schemes 1−3. Kaleidagraph version 4.5 (Synergy

template DNA oligonucleotides for the enzyme strand were from IDT and used without purification. Transcription was from a hemiduplex template using T7 polymerase and performed as previously described.44 Polynucleotide kinase (PNK) was obtained from New England Biolabs (Ipswich, MA). All buffers were prepared using ultrapurified deionized water and filtered with 0.5 μm filters. RNA Sequences and Preparation. The following are the enzyme (E) and substrate (S) strands of the hammerhead ribozyme used, which include the C17U variant. The secondary structure for this ribozyme is provided in Figure 1A. Sequences are from the hammerhead 16 construct, HH16,45 with an additional G at the 5′-end of the enzyme strand to allow for better transcription:45 EHH16 (39 nucleotides), 5′-GGCGAUGACCUGAAGAGGCCGAAAGGCCGAAACGUUCCC; SHH16 (17 nucleotides), 5′-GGGAACGUCGUCGUCGC; S HH16 C17U (17 nucleotides), 5′-GGGAACGUUGUCGUCGC (the sole change is underlined). The enzyme strand of the two-piece minimal hammerhead ribozyme used for these experiments was prepared by in vitro T7 transcription. The substrate RNA strand was 5′-end-labeled with [γ-32P]ATP and polynucleotide kinase and purified by 10% denaturing PAGE. Radiolabeled RNA was crush and soak eluted and ethanol precipitated prior to use in experiments. All kinetic experiments were of the appropriate time scale, going out to 2−4 h, that they could be accomplished with end points between 50 and 95% completion. Reaction Kinetics for the HH16 Ribozyme. Stock solutions of the HH16 enzyme and substrate strands in distilled, deionized water were renatured separately at 90 °C for 2 min and then cooled at room temperature for 10 min before being combined in a solution containing distilled, deionized water and a 10× buffer. The buffer consisted of 50 mM MES (pH 5−6.5), HEPES (pH 7−8.5), CHES (pH 9−10.5), or CAPS (pH 11). To ensure that buffer change was not the cause of the waviness, reactions were also completed in 5 mM Mg2+ at pH 6.5 and 9 using HEPES buffer. No significant changes were found in the observed rate constants using other buffers for reactions. A 3 μL aliquot was removed, mixed with an equal volume of 95% formamide loading buffer and EDTA, and placed directly on dry ice for the zero time point. Ribozyme cleavage was initiated by adding an appropriate volume of a 10× solution of MgCl2 to the remainder of the reaction mixture, mixing vigorously, and incubating at 25 °C. Final concentrations of buffer and KCl were 50 mM each. Reaction progress was monitored by removing 3 μL aliquots at various times, quenching reactions with an equal volume of 95% formamide loading buffer and EDTA, and placing samples directly on dry ice. Aliquots were fractionated via denaturing 15% PAGE. Gels were dried, visualized, and quantified on a PhosphorImager using a cassette with fresh foam backing to ensure sharp bands.46 Plots of fraction cleaved, fc, versus time were fit to either a single-exponential equation (eq 1) or a linearized version of it for very slowly reacting samples that reacted to only ∼50% completion based on the time allotted. Sample fraction cleaved versus time plots are provided in Figure S1. fc = A + Be−kobst

Scheme 1. Two-Dimensional, One-Channel and TwoChannel Noncooperative Models for Ribozyme Cleavagea

a

The key (center) provides the notation for the ribozyme, where the general acid is GA, the general base is GB, and influencers are Inf 1 and Inf 2.

Software, Reading, PA) was used to fit kinetic data to linear or first-order exponential equations. Rate constants as a function of pH, parametric in Mg2+ concentration, were then input into Igor Pro version 6.37 (WaveMetrics, Lake Oswego, OR) to conduct global fitting. All equations are provided in the Supporting Information. The simplest kinetic model is a two-dimensional, onechannel noncooperative model (Scheme 1A). We build from the one-channel model to also include a two-channel noncooperative model (Scheme 1B). Note that the key includes “influencers”, which can affect the population of the functional form of the ribozyme but are not directly involved in catalysis. The derivation of both of these models can be found in the Supporting Information. Under a noncooperative model, there is one pKa and one Kd. Rate−pH kinetic data parametric in Mg2+ concentration were fit globally for linked values of pKa and Kd, allowing for different values of kmax,obs at each Mg2+ concentration (see Figure 3A). The next simplest kinetic model is a two-dimensional, onechannel model that incorporates cooperativity between Mg2+ and proton binding, which is shown in Scheme 2A. Like the noncooperative model, we also incorporate a two-channel cooperative model (Scheme 2B). The derivation of both of these models can be found in the Supporting Information. Under a cooperative model, rate−pH kinetic data parametric in Mg2+ concentration (influencer 1) were fit globally for linked values of pKa2, Kd1, and Kd2, allowing for different values of kmax,obs at each Mg2+ concentration (see Figure 3B). Values for pKa1 were calculated from appropriate thermodynamic relationships as described previously.7 We then considered a three-dimensional, four-channel kinetic model that incorporates cooperativity between binding of a Mg2+ ion (influencer 1) and two protons assigned to G12 and C17 (influencer 2) (Scheme 3). Equations for this model and an accompanying derivation can be found in the Supporting Information. Rate−pH kinetic data parametric in

(1)

The logarithms of the observed rate constants were calculated and plotted against pH. All kinetic reactions were repeated three times, and observed rate constants were plotted C

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Mg2+. A zero time point was removed prior to the addition of the MgCl2 solution. Samples were incubated at 25 °C, and 3 μL aliquots were removed at nine different time points and quenched with an equal volume of 95% formamide loading buffer and EDTA. Samples were loaded onto a denaturing 15% PAGE sequencing gel along with RNase T1 and hydrolysis ladders. Gels were dried, visualized, and analyzed on a PhosphorImager. High-pH ILP gels are provided in Figure S3.

Scheme 2. Two-Dimensional, One-Channel and TwoChannel Cooperative Models for Ribozyme Cleavagea

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RESULTS Application of Global Fitting to the HDV Ribozyme. Rate−pH data are commonly used in characterizing RNA and protein enzymes. Often, a single rate−pH profile is fit with a simple kinetic equation assuming a single ionization and no coupling to other species. More complex data sets that are parametric in another variable, like Mg2+ concentration, require equations that are also parametric in that variable.7 In these instances, secondary (or nested) plots of observed rate constants, such as pKa,obs and kmax,obs, versus Mg2+ concentration become necessary to extract the full complement of intrinsic kinetic parameters. Such secondary kinetic approaches have been used in kinetic studies of the HDV ribozyme.7 These nested plots are prone to larger error, however, and can make statistical evaluation of complex cases difficult. Another approach to evaluating complex data is global fitting, in which all data are fit together to a set of nested variables.48 We were interested in using global fitting as a tool to analyze complex rate−pH data for ribozymes. We benchmarked this approach on our published kinetic data for the HDV ribozyme and then applied it to new data for the hammerhead ribozyme. The HDV ribozyme (Figure 1B) has been extensively studied and displays anticooperativity between the protonated general acid (C75) and a Mg2+ ion in the active site according to Scheme 2A.a,7 We first refit rate−pH profiles individually (i.e., nonglobally) to the same equations previously published and confirmed that the pKa,obs values were identical to the published ones (Figure 2A; values provided in the caption). As expected, the pKa of C75 shifts toward neutrality with the lowering of Mg2+ concentration. Secondary plots of these pKa,obs values versus Mg2+ concentration, according to Nakano et al.,7 led to identical values for the Mg2+-free and Mg2+-saturated ribozyme of 7.25 and 5.9, respectively. We then fit the same kinetic data globally according to Scheme 2A to provide the fits shown in Figure 2B. Through these fittings, we directly obtain the Mg2+-free and Mg2+saturated pKa values (i.e., without the need for secondary plots). The values of Mg2+-free and Mg2+-saturated pKa are 7.2 and 5.9, respectively, which are in excellent agreement with the nonglobal values of 7.25 and 5.9, respectively, mentioned above. Notably, the χ2 values are nearly identical between nonglobal and global fittings, illustrating that global fits to a two-dimensional cooperative model (Scheme 2A) provide a good description of the HDV ribozyme kinetic data. In summary, global fitting provides a simple and robust approach to elucidating kinetic information from rate−pH profiles for small ribozymes without the need for secondary plots. Effects of pH and Mg2+ on the Single-Turnover Reaction for the HH16 Ribozyme. We next turned our attention to the hammerhead ribozyme. Although this was the first ribozyme discovered and has since been the subject of many studies, its mechanism is still unresolved. Recent crystal structures of a native hammerhead ribozyme in three different states (low pH, high pH, and high-pH transition state

a

The key (center) provides the notation for the ribozyme, where the general acid is GA, the general base is GB, and influencers are Inf 1 and Inf 2.

Scheme 3. Three-Dimensional, Four-Channel Cooperative Model for Ribozyme Cleavagea

a

The key (center) provides the notation for the ribozyme, where the general acid is GA, the general base is GB, and influencers are Inf 1 and Inf 2.

Mg2+ concentration were fit globally for linked values of pKa1,G12 and pKa3,G12; pKa1,C17, pKa2,C17, pKa3,C17, and pKa4,C17; Kd1 for Mg2+; and kmax (see Figure 3C). To maximize the accuracy of pKa,C17, all four C17 pKa values were included in this fit; other paths gave similar results (see Figure S2). Values for other variables were calculated from appropriate thermodynamic relationships. In-Line Probing for the HH16 Ribozyme. Hydrolysis of the 32P 5′-end-labeled enzyme alone and bound to a chimeric substrate inhibited by a deoxy at the cleavage site was tracked out to 96 h in the presence of 10 mM Mg2+ at 25 °C using inline probing (ILP).47 The radiolabeled enzyme strand and cold substrate strand (1 μL) were renatured separately in Eppendorf tubes containing 4 μL of 0.5× TE/H2O by being heated at 90 °C for 2 min and then cooled for 10 min back to room temperature. The enzyme and substrate strands (5 μL each) were then combined with distilled, deionized water, 10× reaction buffer [500 mM CHES (pH 9), CHES (pH 10), or CAPS (pH 11)], and 20 mM MgCl2 for final concentrations of 1 nM enzyme, 100 nM substrate, 50 mM buffer, and 2 mM D

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analogue) have reinvigorated the field and provided wellneeded insight into the catalytic mechanism.34,38 Intriguingly, these structures show the absence of a Mg2+ ion (M2) at the Hoogsteen face of putative general base G12 at lower pH values. Given that the minimal hammerhead ribozyme is still highly active in the absence of Mg2+ and presence of excess monovalent ions,49−51 with the rate decreased only ∼10-fold at pH 7.5, a non-Mg2+-derived interaction could influence the deprotonation of the general base. To gain insight into this possibility, we determined rate−pH profiles parametric in Mg2+ concentrations. Several previous studies reported rate−pH profiles for the hammerhead ribozyme;8,9,38,52 while providing insight, these studies were not sufficiently extensive in pH values or Mg2+ concentrations to fully characterize the kinetic profiles. We chose to study a minimal hammerhead construct, HH16, because there is a full kinetic framework established for it and because chemistry has been shown to be rate-limiting under single-turnover conditions.45 Rate−pH profiles for single-turnover reactions of HH16 were collected between pH 6 and 11 at seven different Mg2+ concentrations ranging from 0.5 to 50 mM (Figure 3). These pH values and Mg2+ concentrations were chosen to allow all kinetic constants to be calculated and to afford convenient reaction times for hand mixing. We performed controls that show that these conditions do not lead to appreciable degradation or alkaline denaturation of the ribozyme (see the ILP studies in Supplemental Results and Figure S3 for direct support). Data and fits are provided in Figure 3, and kinetic parameters are listed in Tables 1 and 2. The rate increases with pH, eventually leveling off at pH 9− 10, as previously noted for the hammerhead ribozyme. Inspection of the data between pH 6 and 8 reveals the complexity of the data: the slopes are fractional, being in the

Figure 2. Nonglobal and global fitting of HDV ribozyme rate−pH profiles parametric in Mg2+ concentration. (A) Rate−pH profiles locally fit to eq 6.0 for Mg2+ concentrations of 0.07 mM (black), 0.17 mM (purple), 0.37 mM (blue), 0.87 mM (light blue), 1.9 mM (lime green), 5.0 mM (green), 10 mM (pink), and 50 mM (maroon). Estimated pKa values (not in paretheses) from local fits agree with previously published values6 (in parentheses) of ≥8 (≥8) for 0.07 mM Mg2+, 8.3 ± 0.44 (≥8) for 0.17 mM Mg2+, 7.6 ± 0.2 (7.7) for 0.37 mM Mg2+, 7.1 ± 0.1 (7.1) for 0.87 mM Mg2+, 6.5 ± 0.1 (6.5) for 1.9 mM Mg2+, 6.4 ± 0.1 (6.4) for 5.0 mM Mg2+, 6.1 ± 0.1 (6.1) for 10 mM Mg2+, and 5.8 ± 0.1 (5.8) for 50 mM Mg2+.6 (B) Rate−pH profiles globally fit to eq 2.0. Values for the Mg2+-free and Mg2+-bound pKa values are 7.2 and 5.9, respectively, which agree with local fitting from panel A and previously published values of 7.25 and 5.9, respectively.7 Errors reported in panels A and B are calculated from the errors in the fits and show low noise and high goodness of fit with χ2 values of 0.55 and 0.56, respectively. All data in this figure were previously published.6

Figure 3. Global fitting of HH16 rate−pH profiles parametric in Mg2+ concentration to the noncooperative and cooperative models. Rate−pH profiles globally fit to (A) the noncooperative model (Scheme 1), (B) the two-dimensional cooperative model (Scheme 2), and (C) the threedimensional cooperative model (Scheme 3) using the derivation found in the Supporting Information. All parameters are listed in Tables 1 and 2. χ2 represents the sum of the residuals for all traces. The reported error is calculated from the errors in the global fits and is weighted by the standard deviation between replicate measurements All panels show the same data and differ only by the modeled fits. Concentrations of Mg2+ were as follows: 0.5 mM (black), 1.0 mM (purple), 2.0 mM (blue), 5.0 mM (light blue), 10 mM (lime green), 20 mM (pink), and 50 mM (maroon). Red arrowheads denote G12 pKa values at the lowest and highest Mg2+ concentrations for two-dimensional models. All data in this figure were previously unpublished. E

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Table 1. Comparison of Apparent pKa and Kd Values Globally Fit to the Two-Dimensional, Two-Channel Noncooperative Model (Scheme 1B) and Cooperative Model (Scheme 2B) for HH16a kinetic model

pKa,app (without Mg2+)

pKa,app (with Mg2+)

ΔpKa,app

Kd1,app (mM) (with G12−)

Kd2,app (mM) (with G12H)

χ2

noncooperative (Scheme 1B) cooperative (Scheme 2B)

9.1 ± 0.05 9.4 ± 0.5

9.1 ± 0.05 8.1 ± 0.4

NA 1.3 ± 0.6

9.2 ± 0.3 1.4 ± 1.7

9.2 ± 0.3 33 ± 5

9.82 4.26

a

Comparison of apparent pKa values under noncooperative and cooperative conditions. Under noncooperative conditions, there is no change in the apparent pKa mathematically possible in the presence and absence of a Mg2+ ion. Under cooperative conditions using Scheme 2, the apparent pKa of the putative general base shifts downward one unit from 9.4 to 8.1 in the presence of a bound Mg2+ ion. The apparent pKa cannot be readily assigned to any particular residue because of the complexity of the cubic model, introduced in Table 2, that provides optimal fits. Given that the threedimensional model fits the data much better, all parameters here are considered apparent and given an “app” subscript.

Table 2. pKa and Kd Values for G12, C17, and Mg2+ from the Three-Dimensional Cooperative Model in Scheme 3 for HH16a equilibrium constant pKa,G12 pKa,G12 pKa,G12 pKa,G12 pKa,C17 pKa,C17 pKa,C17 pKa,C17 Kd [1] Kd [2] Kd [3] Kd [4]

[1] [2] [3] [4] [1] [2] [3] [4]

environment

value

C17, with Mg2+ C17H+, with Mg2+ C17, without Mg2+ C17H+, without Mg2+ G12−, with Mg2+ G12H, with Mg2+ G12−, without Mg2+ G12H, without Mg2+ C17, G12− C17H+, G12− C17, G12H C17H+, G12H

9.5 ± 0.5 8.1 9.9 ± 0.3 9.4 9.3 ± 0.8 7.8 ± 0.8 8.5 ± 0.5 8.1 ± 0.4 16.4 ± 3.5 mM 2.9 mM 36 mM 58 mM

Incorporation of cooperativity between Mg2+ and proton binding into the two-dimensional, two-channel kinetic model (Scheme 2B) improved the χ2 to 4.26 but preserved and even accentuated the sinusoidal nature of the residuals (Figure 3B). Of most concern is the fact that the fit curves did not capture, and indeed are mathematically unable of capturing (manuscript in preparation), the wavy nature of the data. The twodimensional cooperative fits provided apparent pKa values of 9.4 ± 0.5 and 8.1 ± 0.4 in the absence and presence of bound Mg2+, respectively, and apparent Kd values for Mg2+ binding of 33 ± 5 and 1.4 ± 1.7 mM, respectivelyc (Table 1). The pKa value in the presence of bound Mg2+ is similar to that recently reported by Mir and colleagues for a native hammerhead ribozyme in the presence of 5.0 mM Mg2+.38 We do not attempt to assign the pKa to any particular residue in these schemes, as the cubic model, introduced below, provides improved fits and pKa assignments. The pKa values listed in Table 1 are thus considered apparent pKa values. To test the validity of global fitting, data sets for each Mg2+ concentration were also fit nonglobally to the two-dimensional cooperative model. Resultant pKa values for each Mg2+ concentration data set are provided in Table S1. Results are in general agreement with the cooperative model, which poses an influence of Mg2+ on the apparent pKa. At the lowest Mg2+ concentration of 0.5 mM, the nonglobal pKa was 9.5 ± 0.1. This value then shifts downward with an increase in Mg2+ concentration, attaining a value of 8.5 ± 0.1 in 50 mM Mg2+. Results from these nonglobal fits are generally consistent with the globally fit parameters for Scheme 2B mentioned above, which determined pKa values ranging from 9.4 ± 0.5 to 8.1 ± 0.4 for the limits of Mg2+-free and Mg2+-saturated sites (Table 1). It appears that the cooperative model describes the data better than the noncooperative model, but that adjustment is needed to obtain fits to the wavy data. Expansion to a Cubic Model To Describe the ActiveSite Environment of the HH16 Ribozyme. The presence of strongly sinusoidal residuals as seen for fits to the kinetic models in Schemes 1B and 2B (Figure 3A,B) is a sign that the data are underfit. We discovered through simulations of rate− pH profiles that cooperativity between one pKa and Mg2+ is mathematically incapable of producing wavy curves like found in the data. However, addition of a second pKa to the models has the mathematical capability of producing such data and fits (manuscript in preparation). Indeed, extension of the twodimensional, two-channel cooperative model in Scheme 2B to a model with a second pKa, which leads to the three-dimensional, four-channel cooperative kinetic model in Scheme 3, significantly improved fitting of the rate−pH data. These fits better capture the shape of the experimental data, the χ2 reduced to 2.90, and the sinusoidal nature of the residuals vanished (Figure 3C). Fits to this three-dimensional model

a

Equilibrium constants and their corresponding determined values from the cubic model. For each of the three species, there are four equilibrium constants related to the protonation state of other nucleobases and availability of a Mg2+ ion. The first column lists the equilibrium constant. The second column lists the protonation state of neighboring nucleobases and whether Mg2+ was in the active site. The third column provides the calculated values based on the derivation found in the Supporting Information. All values listed for each parameter come from the plot fit for all C17 pKa values, as shown in Figure S2. The χ2 value was calculated to be 2.90.

range of 0.6−0.8 when forced to fit to a straight line, and are themselves a function of pH, changing in a nonmonotonic fashion. This leads to “wavy” data, where curves start to level off with pH only to rise again and then level off again. Similarly shaped hammerhead ribozyme rate−pH data have been published previously by several different research groups (Figure S8), although without mechanistic explanation.8,9,38 In all of these published examples, the initial leveling off point occurs around pH ∼8, consistent with our own data. Next, we fit this wavy data set to equations derived from the kinetic models in Schemes 1B, 2B, and 3. Schemes 1B and 2B are twodimensional, two-channel models with cooperativity between Mg2+ and proton binding absent and present, respectively, while Scheme 3 is a three-dimensional, four-channel model with cooperativity among binding of a Mg2+ ion and two protons.39,40,b As shown in Figure 3A, the simple twodimensional, two-channel noncooperative kinetic model from Scheme 1B with parameters for binding of one Mg2+ ion and one proton led to a single pKa of 9.1 ± 0.05 and a Kd for Mg2+ binding of 9.2 ± 0.3 mM, with a very poor fit to the data (Table 1). The residuals from the fits using the equations in the Supporting Information have a χ2 of 9.82 and are sinusoidal, which is indicative of underfitting (Figure 3A). F

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Biochemistry

On the basis of the positioning of M1 and M2 in the crystal structures,34 this metal ion is most likely M2, the metal near the Hoogsteen face of G12 as its binding would be favored most by G12 deprotonation; additionally, M1, the other metal in these crystal structures, is far from the electrostatic pocket between G12 and U6/16.1 (Figure 1C). This notion is supported by the Ca2+ studies (see below); however, we cannot rule out the possibility that there are competing interactions between the two metal ions. Plotting of the rate−pH profiles parametric in Mg2+ concentration (Figure 3) reveals that the maximal observed rate constant, the y value of the plateau, is a function of Mg2+ concentration, making it an observed kmax, or kmax,obs.7 We thus plotted kmax,obs as a function of Mg2+ and found that the resultant curve could be globally fit to a simple saturation curve (Figure S4). We obtained Kd values of 2.1 ± 0.3 and 1.2 ± 0.2 mM for pH 10.5 and 11, respectively, and single linked values for kmax and the Hill coefficient, n, of 0.78 ± 0.04 min−1 and 1.6 ± 0.25, respectively. These data are consistent with a metal ion binding in a saturable fashion at high pH and are in line with Kd values estimated from the two-dimensional and cubic models. Given that the metal in the cube has been assigned to M2 (see above) and the Hill coefficient is 1.6, the Kd likely reflects influencer M2 with a contribution from M1 or other metals. We sought to understand the contribution of various catalytic speciesgeneral acid, general base, their mathematical product, and influencer C17H+to the reaction by deconstructing the rate−pH profiles graphically, akin to earlier approaches from our laboratory.11 We found that wavy data can be captured only if there are two interacting pKa values, with one of the interacting partners, here C17H+, influencing the reaction without itself participating directly in the reaction, i.e., without acting as a general acid or base. This is illustrated in Figure 4 for species plots as a function of pH at different concentrations of Mg2+, where the fraction of putative general base G12− is plotted in blue, the fraction of Mg2+-bound general acid is plotted in red,d and the fraction of fully functional ribozyme (with Mg2+-bound general acid and G12−) is shown as a dashed black line. Overlaid on these plots, in pink, is the fraction of C17 that is protonated, which is wavy and induces a wave in the G12− data through cooperative interaction with G12−. The resultant dashed curves for the fully functional ribozyme are also wavy, as illustrated in Figure 4D; moreover, the wave has a wider intermediate-pH plateau region at higher Mg 2+ concentrations and a narrower intermediate-pH plateau region at lower Mg2+ concentrations like the actual data (Figure 3). When we consider a case in which C17 does not act as an influencer on the pKa of G12 and has a pKa that remains unperturbed at 4.2 independent of the Mg2+ concentration, we observe that the waviness vanishes and the resultant dashed curve no longer mimics the experimental data (Figure S5). We also simulated another variation in which we increased or decreased the rate constant of the protonated C17-containing channels. Increasing the rate constant of these channels enhanced the waviness of the simulations, while decreasing the rate constant diminished the waviness similar to Figure S5. This reinforces the importance of protonated C17 reaction channels to the observed wavy data. Variants of C17U and Mg2+-to-Ca2+ Support the Cubic Model. To test the above mechanism and help assign C17’s role in the mechanism, we sought to make relatively minor changes to C17 and remeasure the rate−pH profiles. Extensive

provided values for the higher pKa ranging from 8.1 to 9.9, for the lower pKa ranging from 7.8 to 9.3, and for the Kd ranging from 2.9 to 58 mM (Table 2). The next step was to assign these values to residues and metal ions. The two pKa values were tentatively assigned to G12 and C17 (influencer 2) and the Kd to M2 (influencer 1). The assignment of G12 to the higher pKa is based on previously published work on the hammerhead ribozyme. Burke and coworkers showed that the leveling off at high pH values corresponded to titration of G12; specifically, they demonstrated that mutagenesis of G12 to 2,6-diaminopurine (diAP), 2-aminopurine (2AP), and inosine affected the apparent pKa by values roughly corresponding to the unshifted pKa differences.9 Several observations support the idea that this pKa belongs to an anionic species. The value of this pKa is shifted most strongly to neutrality, with a value of 8.1, in the presence of the two cationic influencer species: a metal ion and a cationic base (Table 2). Moreover, this value is in good agreement with the pKa of 8.1 ± 0.4 with bound Mg2+ from the two-dimensional cooperative model derived from Scheme 2B (Table 1). Similarly, the value of the high pKa is highest in the absence of both cationic influencer species, at 9.9 ± 0.3 (Table 2), which agrees with the pKa of 9.4 ± 0.5 with no Mg2+ from the two-dimensional cooperative model derived from Scheme 2B (Table 1). Several observations likewise support assignment of the lower pKa to C17. The values of this pKa are most strongly shifted upward in the presence of the anionic G12, at 8.5 ± 0.5 and 9.3 ± 0.8, in the absence and presence of bound Mg2+, respectively (Table 2). Moreover, the rounding points of the intermediate-pH plateau region of the rate−pH profile are farther apart at high Mg2+ concentrations (Figure 3), corresponding well with the wide range of pKa values in the presence of Mg2+, from 7.8 ± 0.8 to 9.3 ± 0.8 (Table 2). Likewise, the rounding points of the intermediate-pH plateau region of the rate−pH profile are closer together at low Mg2+ concentrations (Figure 3), corresponding well with the narrow range of pKa values in the absence Mg2+, from 8.1 ± 0.4 to 8.5 ± 0.5 (Table 2). Because C17 stacks directly under G12 (Figure 1C), we assigned this pKa to C17, an assignment that is supported by C17U and Mg2+-to-Ca2+ variant effects on the rate−pH profile (described in the next section). The cubic model contains 12 unknowns, corresponding to each edge of the cube in Scheme 3. Seven parameters were floated in the cubic model, while the other five were calculated using equilibrium relations. The robustness of these calculations, supported in part by the reduction in χ2 mentioned above and by the reduction in the sinusoidal nature of the residuals, was evaluated by taking three different paths along the cube, which allowed a different seven-member subset of the 12 parameters to be directly calculated. Values of the 12 parameters were in good agreement with each other independent of the path taken (Figure S2). For instance, all three evaluations revealed that the pKa of G12 is shifted closest to neutrality in the presence of a cationic C17H+ and a Mg2+ ion. Moreover, the pKa of G12 was estimated to be the highest in the absence of protonated C17 and a Mg2+ ion. Likewise, under all three evaluations, C17 is estimated to have a shifted pKa over ∼3.5−5 units toward neutrality depending on the environment, denoting the importance of the protonated form for electrostatic stabilization. Lastly, all three evaluations showed that a Mg2+ ion bound most tightly in the presence of C17+ and G12− and most weakly when G12 was protonated. G

DOI: 10.1021/acs.biochem.7b00174 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

while the N3 and N4 positions are not as important, with krel effects of only ∼5-fold. In the hopes of obtaining mechanistic information about position 17 without destroying the active site, we replaced C17 with U, which maintains the critical O2. Notably, U differs from C in that U is neutral in the N3protonated form and anionic in the N3-deprotonated form. We conducted rate−pH experiments for C17U and wild-type HH16 ribozymes at both 10 and 20 mM Mg2+ (Figure S6). Overall, the C17U variant behaves like the wild type but with a decrease in rate of ∼7-fold in the range of pH 6−8, consistent with previous reports (Table 3), and a decrease of 10−15-fold at higher pH values. Interestingly, the C17U data preserve and even accentuate the wavy data found for the wild-type ribozyme (Figure S6). One possibility is that at pH 8, the U is ionizing to its anionic form, which may either “lure” M2 away from G12 and misposition it for catalysis or disfavor G12 deprotonation to prevent proximal anionic charges. Indeed, the imino nitrogen atoms of G12 and N3 of residue 17 are only 4.0 Å apart. Overall, the rate−pH profiles for the C17U variants are consistent with the cubic model and assignments provided in Scheme 3. To test our mechanism further, we determined the effect of changing the divalent metal ion from Mg2+ to Ca2+ on the rate−pH profiles (Figure S7). Studies by Herschlag and coworkers supported a single metal ion with ligands to the pro-RP oxygen atoms of the scissile phosphate and A9 phosphate as well as N7 of G10.1.50,53 The Herschlag study is supported by extensive phosphorothioate experiments (Table 3) and has been corroborated by the recent crystal structures of Mir and Golden that show a metal ion (=M1) with some characteristics of this one, albeit with distances to the pro-RP oxygen of the scissile phosphate on the long side (ground state) or to the proSP oxygen instead (transition state analogue).34 Rate−pH data for the wild-type ribozyme in 10 mM Mg2+ or 10 mM Ca2+ are provided in Figure S7. Notably, the Ca2+ rate− pH profile lacks the wavy nature of the Mg2+ rate−pH profiles. The slope of the 10 mM Ca2+ data is unity, and fits to the 10 mM Ca2+ data with the standard rate−pH equation11 have an excellent χ2 of 0.20, as compared to a χ2 of 0.31 for the 10 mM Mg2+ profile fit to this standard equation instead of the wavy curve described above. In addition, the rate−pH profiles for the Ca2+ data almost “catch up” to the Mg2+ data at high pH: at low pH, rates are ∼2 log slower in 10 mM Ca2+ than in 10 mM Mg2+ but at high pH are only ∼1 log slower. The “catch-up” nature of these data is consistent with M1, the site filled in lowand high-pH crystal structures, as being Mg2+-specific, but M2, the site filled only in the high-pH structures, being promiscuous. The specific and promiscuous nature of these sites was first pointed out by Herschlag and colleagues.53

Figure 4. Species plots of various catalytic species for the hammerhead ribozyme. Data are plotted as a function of pH at three Mg2+ concentrations for the three-dimensional cooperative model: (A) low Mg2+ (0.5 mM), (B) intermediate (physiological) Mg2+ (2.0 mM), and (C) high Mg2+ (20 mM) concentrations. (D) Comparison of the fraction of the functional form of the ribozyme with Mg2+. Blue traces represent the sums of the species containing the functional form of the putative general base, f B− (G12−) [(1) + (4) + (5) + (8)]. Red traces represent the populations of the functional form of the general acid associated with Mg2+, f HA−Mg2+. Pink traces represent the sums of the species containing the protonated C17H+ [(3) + (4) + (7) + (8)], which impacts the population of G12− but is not modeled to directly transfer protons. The dashed line represents the product of the fraction of the functional forms of the general base and Mg2+associated acid ( f B− × f HA−Mg2+) and represents the observed rate−pH profiles. As discussed in the Supporting Information, additivity is a reasonable assumption given that the general acid has a high pKa. The pale blue vertical rectangles show the portions of the rate−pH profiles that are accessible by experiments without acid or alkali denaturation of the ribozyme. Simulations were prepared using constants from fits to experimental data herein.

studies of C17 variants of the HH16 ribozyme at neutral pH have already been published, as summarized in Table 3. The O2 position of C17 is known to be particularly important for catalysis, with krel effects of more than 500-fold, Table 3. Published Mutational Studies of HH16 U6 and C17a residue

functional group

modification

krel

U6/16.1

N3/N4 O2′ O2 N3 N4 O2′ pro-R-O pro-S-O

cytidine 2′-deoxy 2-pyr, 4-pyr, and U uridine 2-pyr 2′-deoxy Rp-phosphorothioate Sp-phosphorothioate