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Biochemistry
 
Biochemistry. 2011 April 12; 50(14): 3004–3013.
Published online 2011 March 18. doi:  10.1021/bi1018336
PMCID: PMC3071035

The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

Abstract

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Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ~1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

Site-specific modification of folded RNA by enzymes is common to many biological processes. The molecular bases of these processes are not understood sufficiently because of the dual requirements for structural analyses that identify candidate surface contacts and for solution studies that quantitatively evaluate their contribution to site-specific cleavage. The site-specific ribotoxin restrictocin is a suitable candidate for such studies because it is well characterized both structurally1,2 and kinetically.3,4

Restrictocin and related fungal ribotoxins such as α-sarcin are small endoribonucleases that cleave one site in the conserved sarcin/ricin loop (SRL) in 23S-28S rRNA to disrupt GTPase activation when elongation factors bind to the ribosome,(5) halt protein synthesis, and ultimately trigger apoptotic cell death (reviewed in refs (6) and (7)). Ribotoxins share the same fold and catalytic mechanism as the well-studied T1 endoribonuclease family of fungal enzymes.1,8,9 In these enzymes, in-line attack of a nucleophilic 2′-hydroxyl group on the 3′ adjacent phosphate produces a 5′-hydoxyl group and a 2′,3′-cyclic phosphate3,4 (Figure (Figure1).1). Despite these similarities, substrate specificity differs greatly. T1 enzymes cleave on the 3′-side of every G nucleotide in single-stranded RNA. In contrast, ribotoxins recognize a single-folded RNA structure on the ribosomal surface, the SRL, and specifically cleave a single phosphodiester bond within the SRL tetraloop. For structural and functional studies, including those described herein, a minimal substrate is used in which the ribosome is trimmed to the ~30 nucleotides of the SRL sequence, designated the SRL substrate.7,10 This minimal substrate is sufficient to replicate the site-cleavage observed in the SRL embedded in the ribosome.(11) Moreover, restrictocin catalyzes the cleavage of this minimal substrate and the ribosome with the same kcat value.(3)

Figure 1
Kinetic and structural models for SRL substrate recognition and cleavage by restrictocin. (A) Reaction scheme for restrictocin (E) and its substrate the SRL (S). The single-turnover kinetic parameter K1/2 monitors initial binding to form the E:S complex ...

Structural studies identified distinctive features in the SRL RNA that contribute to site-recognition by ribotoxins. The loop portion of the SRL stem-loop structure folds into two motifs: a GAGA tetraloop and a bulged-G motif1216 (Figure (Figure1).1). Both motifs contribute to ribotoxin recognition,4,17 with cleavage occurring between the third and the fourth nucleotide of the tetraloop (Figure (Figure2).2). To identify the enzyme substrate interface, co-crystal structures were determined for restrictocin bound to several different substrate analogues.(2) These structures provide the basis for a model that involves simultaneous docking of the protein to both SRL motifs (Materials and Methods and Figure Figure1C,D).1C,D). A loop containing K110, K111, and K113, designated the lysine triad, recognizes the bulged-G nucleobase of the eponymous motif and the enlarged major groove of the SRL. A β-sheet surface recognizes a base flipped form of the target nucleotide (the second G of the GAGA tetraloop) that places it in the active site with an in-line orientation poised for cleavage.

Figure 2
Substrates used in this study. The secondary structures of the SRL (reproducing nucleotides 4311−4337 of 28S rRNA from Rattus norvegicus), 7dN, tetraloop, and ssNA substrates with the tetraloops, bulged-G motifs, and stem structures highlighted ...

From the RNA side of the enzyme−substrate interface, mutations in the tetraloop and stem are tolerated. In contrast, mutations of the bulged-G nucleobase to A, C, or U abolished detectable SRL cleavage, providing evidence that the bulged-G motif is an important element for SRL recognition.(17) It is unclear, however, whether this loss of activity arises from disruption of protein contact(s) to the bulged-G nucleobase or from indirect effects. Mutation of this bulged-G either destabilizes the motif because in the absence of SRL structural rearrangements neither an A, C, nor U mutation can replace the stabilizing contacts formed by the bulged-G nucleobase or changes the SRL structure to restore these stabilizing contacts. From the protein side of the enzyme−substrate interface, structural studies,1,2 kinetic analyses of point mutants,(9) and similarity to the sequence and 3D structure of T1 nucleases have implicated active site residues H136 and E95 as the general acid and base, respectively.(9) Even though Y47, H49, and R120 are expected to stabilize the transition state,9,1820 their function is less well established. A nonsystematic survey of mutants identified two residues that affect specificity based on qualitative assays of cleavage of the SRL and/or the ribosome: Y47 and H49 in the active site.19,21 A systematic deletion study, which again assayed cleavage qualitatively, indicated that the activity also depends on the lysine triad that is near but outside of the active site.(22)

As a prerequisite to determine the extent to which, and when during the reaction, each interface contact contributes to recognition, we previously established a kinetic framework for analysis of the reaction.3,4 The reaction proceeds via two steps (Figure (Figure1A).1A). The first step, involving formation of an electrostatic complex (E:S), is monitored by Km or the single turnover equivalent K1/2, which are both equal to the KD for binding of enzyme and substrate.3,4 The second step, involving site recognition (hereafter designated docking) and RNA cleavage, is monitored by the catalytic constant kcat (or the single turnover equivalent k2). Restrictocin cleaves the SRL ~1000-fold faster than a single-stranded RNA noncognate substrate, providing an empirical definition of the basal specificity.3,4 Because formation of the E:S complex is nonspecific,3,4 specificity is achieved during the subsequent kcat (or k2) step.

Here, we exploited this kinetic framework to systematically investigate which contacts in the enzyme substrate interface contribute to specific recognition and determine the relative contribution that each makes to the specificity. This was done by constructing restrictocin point mutants that selectively disrupt interface contacts and comparing k2 values for cleavage of the SRL substrate relative to a single-stranded substrate. To investigate which RNA structural elements contact protein interface residues, k2 values were compared for restrictocin, or mutants thereof, cleaving variant substrates in which all or part of the SRL motifs have been removed. Our findings show that, as with other interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximal catalytic activity occurs only when both SRL motifs are intact.

Materials and Methods

Expression and Purification

Point mutants of restrictocin (D40A, K42A, Y47F, H49A, H49Q, F51A, T52A, R65A, K110A, K111A, K113A, K113R, K113Q, Q141A, D143A) were made with QuikChange (Stratagene) by using the pREST plasmid,(23) which contains the wild type (Wt) ribotoxin gene from Aspergillus restrictus, as the template. Wt and mutant restrictocin were recombinantly expressed, purified, and stored as described before.4,23

Construction of a Structural Model of the SRL−Restrictocin Complex

Even though this model has been previously cited (Figure (Figure11B),(2) the details of how it was built were not described and therefore are presented here. Except for one nucleotide, the model was built from fragments of structures determined by X-ray crystallography and demonstrates that recognition of the bulged-G motif and site-specific cleavage can occur simultaneously. The largest fragment is from the “bound” structure(2) and includes coordinates for restrictocin and the SRL RNA, but excludes the GAGA tetraloop of this RNA. Coordinates for the first G and final A in the tetraloop are from the uncomplexed SRL structure(24) based on superposition with the bulged-G motif and stem from the bound structure. The uncomplexed structure is expected to reflect the ground state structure. Coordinates for the cleavage site nucleotide (second G of the tetraloop) were defined by superposition of restrictocin with the related RNase T1 structure, in which T1 was co-crystallized with a noncleavable dinucleotide substrate that shows how the target G docks in the active site.(25) Lastly, the A 5′ adjacent to the target G (the first A in the GAGA tetraloop) was modeled, followed by energy minimization to ensure reasonable stereochemistry.

RNA Substrates

Oligonucleotides, herein designated SRL, 7dN, tetraloop, and ssNA substrates (Figure (Figure2)2) and the 2′-OMe substrate (Table (Table2),2), were synthesized either by Dharmacon, Inc. or at the University of Chicago. Three were purified by nondenaturing 20% 29:1 PAGE: the SRL substrate (5′-CCU GCU CAG UAC GAG AGG AAC CGC AGG), the 2′-OMe substrate (5′-CCU GCU CAG UAC GAX AGG AAC CGC AGG, where X is a 2′-methoxy substituted G), and the 7dN substrate (5′-CCU GCU CAX UAC GAG AGG AAC CGC AGG, where X is a 7-deazaguanosine nucleotide). The SRL substrate reproduces the nucleotides at positions 4311−4337 of 28S rRNA from Rattus norvegicus. The other two were purified by denaturing 20% 29:1 PAGE: the tetraloop substrate (5′-GCG GUU CCG AGA GGA ACC GC) and the ssNA (single-stranded nucleic acid) substrate (5′-d(TGGTAAT)-G-d(AGCTGACGGACAT)) designed to lack secondary structure.(26) Nondenaturing purification was used to remove a population of slow-cleaving RNA molecules from the substrate sample, which comigrates with the active substrates on denaturing PAGE.(4) These inactive species were not observed in tetraloop and ssNA substrates.

Table 2
Comparison of Catalytic Constants (kcat or k2 (s−1)) for Various Substratesa

Kinetic Assays

Cleavage of the 32P-labeled oligonucleotide substrate was performed as before(4) in 10 mM Tris (pH 7.5) containing 0.05% (v/v) Triton X-100 at 37 °C under k2 conditions (S0 [double less-than sign] K1/2 [double less-than sign] E0) or under k2/K1/2 conditions (S0 [double less-than sign] E0 [double less-than sign] K1/2). To avoid the use of rapid stopped flow quench kinetics and maintain the same measuring technique for all reactions studies, kcat was measured instead of k2 for the Wt enzyme and mutants with near Wt activity with S0 [dbl greater-than sign] Km [dbl greater-than sign] E0. After initiating the reaction by rapid addition of 5′-[32P]-labeled oligonucleotide substrates, 1.5 μL aliquots of the reaction were removed during a reaction time course and quenched by addition of 3.5 μL of stop solution (10 M urea containing 1% (w/v) SDS, 0.02% (w/v) xylene cyanol, and 0.04% (w/v) bromophenol blue). Samples were separated using 20% 29:1 PAGE containing 0.5 × TBE (45 mM Tris-borate (pH 8.3) and 1 mM EDTA) and 7 M urea. Samples were quantified using a PhosporImager (Molecular Dynamics). The time-dependent loss of substrate was fit to eq 1 using least-squares regression procedures in Origin (Microcal):

equation image

where FracS is the fraction of substrate, A is the cleavable fraction of the substrate, kobs is the observed rate as defined in Figure Figure1A,1A, t is time, and y0 is the noncleavable fraction of the substrate. Under conditions of saturating enzyme (Eo [dbl greater-than sign] K1/2) kobs = k2 and under conditions of saturating substrate (So [dbl greater-than sign] K1/2) kobs = (k2Eo)/K1/2. To ensure a reasonable fit, the sum of substrate fractions (A + y0) was set to a value of 1.

We exploited the equivalence of kcat and k2(4) to efficiently measure rate constants for Wt and mutant enzymes. For Wt restrictocin, and mutants with near-Wt rates between 0.1 and ~1 s−1, k2 cannot be determined without the use of a rapid mixing and quenching techniques. On the other hand, these rate constants can be readily measured under kcat conditions ([S0] [dbl greater-than sign] [E0]). Conversely, for mutants with slow cleavage rates (<~0.1 s−1), kcat cannot be readily measured due to the long time-points and high substrate concentrations required to observe cleavage. Thus, slow rates were determined under k2 conditions ([S0] [double less-than sign] [E0]).

Experimental Errors

Reported values represent the average and standard deviation of at least three measurements. All reported differences are statistically significant, with >90% confidence in a Student’s t test.

Results

Designs to Disrupt the Enzyme−Substrate Interface

To probe the protein side of the enzyme−substrate interface, we mutated 12 residues located at the crystallographically determined interface (Figure (Figure1B,1B, magenta surface). Protein backbone contributions were not evaluated (Figure (Figure1A,1A, blue surface). Of the 10 side chains that contact the RNA substrate (<3.4 Å), scanning alanine mutagenesis was performed on 9: K42A, H49A, T52A, R65A, K110A, K111A, K113A, Q141A, and D143A. For the 10th, we mutated Y47 to phenylalanine instead of alanine to disrupt a putative contact to the scissile phosphate (Figure (Figure1C)1C) while maintaining the capacity to form other active site contacts observed in the structure. The 11th mutant, D40A, is expected to disrupt binding of a potassium ion, which also contacts the SRL tetraloop.(2) The 12th mutant, F51A, is located in the active site pocket but does not contact the substrate (>3.4 Å). Mutations of other active site residues, which include R120, the putative general base, E95, and putative general acid, H136, were not analyzed herein because their contributions to catalysis have been characterized previously.9,1820,2730

To investigate the contribution of specific restrictocin−SRL contacts from the RNA side, variants that included multiple mutations were used rather than point mutants. The tetraloop and bulged G motif each form non-Watson Crick interactions.1216 Currently, it is impossible to predict how mutations in these motifs will affect the SRL structure. Thus, mutations of non-Watson−Crick interaction in either SRL motif were avoided to prevent unpredictable perturbations to the RNA structure. Four RNA variants were used. One is missing the putative N7 contact to the bulged-G (7dN substrate), a second is a hairpin containing the tetraloop motif but lacking the bulged-G motif (tetraloop substrate), and a third is an unstructured single-stranded nucleic acid (ssNA substrate) (Figure (Figure2).2). The fourth one is the SRL with site-specific cleavage blocked by replacing the nucleophilic hydroxyl group with a methoxy group (2′-OMe substrate in Table Table2;2; Materials and Methods).

Contacts Important for the Rate of SRL Cleavage by Restrictocin

To differentiate mutations that affect E:S stability from those that affect docking and cleavage, we determined ratios of Wt to mutant k2/K1/2 and k2 (Materials and Methods; Tables Tables11 and and2).2). K1/2 monitors nonspecific equilibrium association of E and S to form the E:S complex and k2 monitors subsequent docking and cleavage (Figure (Figure1A).1A). Ratios of Wt to mutant k2 values are designated by krel and the substrate used: krel (SRL), krel (ssNA), krel (7dN), and krel (tetraloop). Ratios based on k2/K1/2 are specifically indicated. The k2/K1/2 and k2 ratios follow similar trends suggesting that changes to k2/K1/2 reflect k2 contributions to docking and cleavage, not to formation of the E:S complex (Figure (Figure3A,3A, compare gray bars to open bars). Therefore, only k2 ratios will be discussed hereafter.

Figure 3
Effect of restrictocin mutations on cleavage of the SRL (A), ssNA (B), 7dN (C), and tetraloop (D) substrates. For each substrate the krel (k2 (Wt)/k2 (mutant)) is shown for each protein mutant(gray bars) and color-coded onto the protein surface (right) ...
Table 1
Mutant Kinetic Parameters (k2/K1/2 (M−1 s−1))a

The krel (SRL) values for restrictocin mutants (Table (Table11 and Figure Figure3)3) range from a high of 2190 ± 150 to a low of 1 (Figure (Figure3A),3A), indicating that the surface residues contribute differentially to k2 and therefore affect either substrate docking, activity or both. Mutation of four contact residues (D40A, K42A, T52A, and Q141A) located at the periphery of the interface had krel (SRL) < 10, reflecting small energy differences of less than 1.4 kcal mol−1. Evidently, these disrupted contacts make minor contributions to substrate docking and activity. The remaining eight mutations (Y47F, H49A, F51A, R65A, K110A, K111A, K113A, and D143A) have krel (SRL) ≥ 10 and cluster along the RNA helical axis of the enzyme−substrate interface.

The largest effects, krel (SRL) > 1000, are seen with H49A and K113A. The effect of the K113A mutation is in accord with structural studies(2) showing that this lysine residue forms sequence-specific interactions with the SRL by contacting N7 and O6 of the bulged G (Figure (Figure1D).1D). To assess the contributions of either hydrogen bonding or salt bridge interactions made by the K113 amino group, two mutants were made. Mutation to K113R partially restores k2, whereas K113Q offers no restoration, with a krel (SRL) > 1000 like K113A. These results provide evidence that the positive charge at residue 113 contributes to this interface contact.

Unlike K113, H49 is in the active site and based on kinetic studies(9) is expected to contribute to transition state stabilization. This contribution may arise from charge neutralization of the transition state by H49, which is expected to carry partial positive charge under the reaction conditions based upon the pKa value of 7.7 ± 0.2 measured for the free enzyme(29) (substrate binding is expected to perturb the pKa further). Also, the imidizoyl moiety of H49 contacts the oxygen atom of the nucleophilic 2′ hydroxyl group in the structural model (Figure (Figure11B)(2) and in the structure of RNase T1 bound to a substrate analogue.2,25 This contact may help nucleophilic activation by the putative general base (E95). The H49Q mutation, expected to maintain hydrogen-bonding capabilities, restores only 16-fold of the 2190-fold k2 loss for krel (SRL) observed for the H49A mutant. The remaining 131 ± 66 fold for krel (SRL) of H49Q may arise from the loss of electrostatic catalysis provided by the partial charge of H49 contributing to electrostatic catalysis29,31 or from other factors.

Y47F and K111A showed noteworthy effects with krel (SRL) values of 280 ± 60 and 110 ± 20, respectively. On the basis of mutational(21) and crystallographic studies(2) (Figure (Figure1C),1C), the active site residue Y47 contributes to transition state stabilization. We examined Y47F to test the contribution of the putative contact between its hydroxyl group and the scissile phosphate (Figure (Figure1C).1C). Positioned outside but near the active site, K111 is observed to contact the phosphodiester backbone of the bulged-G motif (Figure (Figure1D),1D), in contrast to K113, which contacts the bulged-G nucleobase. Loss of activity for mutants of these residues suggests that the hydroxyl group of Y47 and the backbone contacts made by K111 contribute to site-specific cleavage.

Four mutants, R65A, F51A, K110A, and D143A, had a smaller effect on krel (SRL), with ratios between 10 and 100. F51 is located in the pocket for binding the target nucleobase but does not contact the substrate; perhaps mutation of this residue affects k2 values by perturbing the active site structure. R65 is expected to form a salt bridge to a phosphate group in the SRL tetraloop; the loss of activity when removing this contact implies that contacts to the tetraloop contribute to SRL recognition (see the krel (tetraloop) data below). D143 lies at the edge of the SRL tetraloop binding site and may therefore contribute directly or indirectly to SRL recognition. Like K111 and K113, K110 contacts to the bulged-G motif (Figure (Figure1D)1D) contribute to recognition.

Differential Roles of Contacts That Contribute to Specificity and Basal Activity

The krel (SRL) data in Figure Figure3A3A do not differentiate between effects on specificity and on basal endoribonuclease activity. The latter does not contribute to specificity because loss of basal activity indiscriminately slows cleavage of all substrates. Because the initial binding step between restrictocin and the SRL substrate is nonspecific,3,4 specificity is defined herein as the ratio of k2 for specific SRL cleavage vs cleavage of the unstructured ssNA substrate. Basal activity is defined as cleavage of a substrate mimic that shares the same rate-limiting step as cleavage of the SRL substrate but lacks site-specific recognition opportunities. Cleavage of the ssNA substrate by restrictocin approximates basal activity because this substrate satisfies five design criteria. First, it is 21-nucleotides long to ensure that the stability of its initial nonspecific electrostatic E:S complex (Figure (Figure1A,1A, step 1) is indistinguishable from that of the SRL substrate.3,4 Second, to simplify cleavage rate measurements we made a single cleavage site by including only one ribonucleotide. Third, the scissile phosphate is flanked on both sides by single-stranded deoxynucleotides to disrupt contacts that recognize features of a specific SRL fold. Fourth, the scissile phosphate of both the ssNA and SRL substrates are flanked by G and A bases to permit them to dock in a similar manner when bound to restrictocin. Fifth, a related single-stranded RNA substrate and the SRL substrate have similar pH−rate profiles, suggesting that these substrates have the same rate-limiting step, and the k2 values for restrictocin cleavage of the ssNA and ssRNA substrates are identical.(26) Therefore, we infer that the ssNA and SRL substrates have the same rate-limiting step.

Under our assay conditions, k2 for ssNA is ~1000 smaller than k2 for the SRL substrate but still ~1 million fold larger than the rate constant for the uncatalyzed reaction (estimated using equation e from ref (32)). Assuming a single mode of docking as shown in Figure Figure1C,1C, catalytic residues like Y47 and H49, which do not make sequence specific contacts, would be expected to affect both basal activity and specific cleavage. To test whether the krel (SRL) effects are due to loss of basal activity we determined k2 for each mutant using the ssNA substrate.

Mutation of active site residues has only minor effects on basal activity, krel (ssNA) < 10 (Table (Table11 and Figure Figure3B).3B). Similar results were observed for the other mutants, except for K113A, which had a slightly larger value of 18 ± 5. Mutation of K113 to either arginine or glutamine attenuates this effect (reducing it to <10). Perhaps K113 assists in the docking of both the SRL and nonspecific substrates, whereas K113R and K113Q assist in the docking of only the SRL substrate.

Quantitation of Cleavage Specificity

To directly investigate contributions of each interface contact to specificity, we determined for each mutant the k2 ratio for cleavage of the SRL RNA and the nonspecific ssNA substrate (Figure (Figure4).4). Only one alanine mutation abolishes specificity by dropping this k2 ratio by over 1000-fold from 955 to 0.8 (H49A). Y47F, K110A, K111A, and K113A also show notable effects, reducing specificity by 96-, 38-, 115-, and 57-fold, respectively. Smaller effects, within ~10-fold of the k2 Wt ratio, are observed for D40A, K42A, F51A, T52A, R65A, Q141A, and D143A.

Figure 4
Effect of restrictocin mutation on SRL specificity. Specificity is defined as the ratio k2 (SRL)/k2 (ssNA) with values >100 (green), from 10 to 100 (yellow), from 1 to 10 (orange), and from 0 to 1 (red). Error bars and markings are as in Figure ...

The observation that the active site mutants Y47F and H49A greatly reduce SRL cleavage (Figure (Figure4)4) without affecting basal activity (krel (ssNA) < 10 and Table Table22 and Figure Figure3B)3B) indicates that Y47 and H49 participate in SRL cleavage but not the ssNA cleavage. The finding that the SRL substrate, but not the ssNA substrate, engages Y47 and H49 in the cleavage mechanism implies that these substrates dock differently into the active site (Figure (Figure55).

Figure 5
The mode of substrate docking determines its cleavage rate. All nucleic acid substrates (S) with a length greater than about 20 nucleotides bind to restrictocin (E) with similar affinities and form an electrostatic complex, E:S. Specificity is conferred ...

To investigate the contribution of interface residues to cleavage fidelity (the preference for cleavage at the correct site versus other sites within the SRL), we monitored the rate of cleavage for the 2′-OMe substrate methylated to block cleavage at the correct site but permit cleavage at other sites within the SRL (Figure (Figure2).2). For the four mutants tested (D40A, K110A, K111A, and K113A; Table Table2),2), the average rate of miscleavage (overall k2 divided by the number of miscleavage sites within the methylated SRL substrate) was comparable (<10-fold difference) to k2 for the ssNA substrate. These results suggest that the same restrictocin residues that contribute to SRL cleavage specificity also contribute to cleavage fidelity and that in the context of the SRL structure restrictocin residues impart their maximal catalytic contributions only during cleavage at the correct site. Like the ssNA substrate, the 2′-OMe substrate does not fully engage the catalytic machinery, supporting the notion that the enzyme−substrate complex populates a distinct nonspecific docking mode during miscleavage. In subsequent experiments, we investigated how the bulged-G motif contributes to specificity by determining krel for the substrates that either partially or completely removed the bulged-G motif (7dN and tetraloop substrates, respectively) and compared these values to krel (SRL).

Recognition of the Bulged-G Motif by Restrictocin

In the structural model (Figure (Figure1D),1D), K113 resides within hydrogen bonding distance of the N7 and O7 atoms of the bulged-G nucleobase in the eponymous motif. To probe recognition of this nucleobase, we disrupted the contact between K113 and N7 by using the 7dN substrate in which the bulged-G nucleobase is replaced by a 7-deazaguanosine (Figure (Figure2).2). This single-atom substitution is nondisruptive of the SRL fold(16) and thus offers an appealing way to test the importance of N7 contacts. The 7dN modification reduces k2 ~ 60-fold for wild-type restrictocin, eliminating a large fraction of the ~1000-fold specificity. This finding implies that interaction with N7 of the bulged-G by restrictocin contributes to catalysis and specificity, in accord with the structural model (Figure (Figure11D).

We also determined the contribution of individual restrictocin residues to recognition of the 7dN substrate (Table (Table11 and Figure Figure3C).3C). The krel (7dN) ratios range from a high of ~190 to a low of 1. Six of the mutations show a minor effect (krel (7dN) < 10): D40A, K42A, T52A, R65A, Q141A, and D143A. Five mutations showed larger effects (krel (7dN) > 10). Three of these (Y47F, H49A, and F51A) are in the active site, whereas the remaining two (K110A and K111A) contact the bulged-G motif (Figure (Figure1C,D).1C,D). The largest change, krel (7dN) > 100, was observed for K113A, which is unable to contact either O6 or N7 of a bulged-G nucleobase. The N7, not the O6, contact is expected to be disrupted by the deaza modification of this substrate variant. To assess the role of hydrogen bonding and salt bridge contacts in SRL recognition by K113, the arginine and glutamine mutants were tested. The K113R mutant restores k2 activity to within ~3-fold of the Wt level, whereas the K113Q does not. These results provide evidence that electrostatic interactions between the cationic K113 and the anionic RNA backbone contribute to the docking and catalytic step (k2). Apparently, K113 is involved in both hydrogen binding and electrostatic interactions with the SRL substrate.

Tetraloop Recognition by Restrictocin

Our previous studies showed that the tetraloop substrate is cleaved specifically.(4) To identify restrictocin contacts that contribute to tetraloop recognition, we determined krel (tetraloop) (Table (Table11 and Figure Figure3D).3D). Like the 7dN substrate, the tetraloop substrate is cleaved ~60-fold more slowly than the SRL by Wt restrictocin. Five mutants (D40A, H49A, T52A, Q141A, and D143A) had only minor effects on activity (krel (tetraloop) < 10), indicating that these side chains do not make notable contributions to tetraloop recognition in the absence of the bulged-G motif. The remaining six mutants had krel (tetraloop) >10-fold: Y47F, F51A, R65A, K110A, K111A, and K113A. With the exception of R65A, these mutants also had reduced activity on the 7dN substrate. Interestingly, R65 contributes more to recognition of the tetraloop substrate than it contributes to recognition of the SRL substrate: the R65A mutant has the largest krel (tetraloop), with a value of 103 ± 26 (Figure (Figure3D),3D), whereas its krel (SRL) is only 9.5 ± 1.8. Moreover, the R65A mutant has a k2 ratio for cleavage of the SRL RNA vs the nonspecific ssNA substrate that is ~10-fold less than this ratio for Wt enzyme (Table (Table22 and Figure Figure44).

Loss of tetraloop substrate activity for the K110A, K111A, and K113A mutants was unexpected because these mutations delete putative contacts to the bulged-G motif, which has been removed from the tetraloop substrate. A plausible explanation arises from a simple modeling study (data not shown) that replaces the bulged-G motif with an A-form stem structure. In the model, the trajectory of the phosphate backbone of the A-form stem structure in the tetraloop substrate approaches close enough to form electrostatic interactions with K110, K111, and K113. To further test the contribution of K113, krel (tetraloop) values were determined for the arginine and glutamine mutants. K113R restores tetraloop substrate activity to Wt levels, whereas K113Q does not. These findings offer evidence that K113, and perhaps K110 and K111 as well, contribute to docking via electrostatic interactions to the WC stem of the tetraloop substrate.

Interestingly, H49A shows no krel (tetraloop) effect (Figure (Figure3D),3D), whereas it shows a medium effect for krel (7dN) (Figure (Figure3C)3C) and large effects for both krel (SRL) (Figure (Figure3A)3A) and specificity (Figure (Figure4).4). An H49 contribution to k2 for reactions with the SRL and the 7dN substrates, but not with the tetraloop and ssNA substrates, suggests that bulged-G motif recognition is required to engage H49 in catalysis. Thus, our findings show that although contacts to the tetraloop are sufficient to produce specific cleavage, maximal cleavage rates and specificity are observed only when the enzyme contacts both SRL motifs: the bulged-G motif and tetraloop.

Discussion

The findings reported here systematically probe ribotoxin residues and the extent to which each one contributes to substrate recognition. Scanning mutagenesis reveals that of the 12 residues predicted to lie within the RNA−protein interface, only 7 contribute to activity (Tables (Tables11 and and2;2; Figures Figures33 and and4).4). Of these, only mutation of the active site H49 (Figure (Figure1C)1C) completely abolishes the specificity of restrictocin by reducing its rate of cleavage to that of the nonspecific ssNA substrate (Figure (Figure4).4). Four other residues make notable contributions to enzyme recognition of the SRL RNA. Y47 is in the active site. In contrast, the other three residues (K110, K111, and K113) are located outside of the active site and recognize the bulged-G motif. As with two other well-studied RNA−protein interfaces, the signal recognition particle and MS2 coat protein,33,34 only a small subset of the interface contacts, often designated “hot spots”, contribute to recognition. The view emerging from these studies and studies of protein−protein interfaces(35) is that hot spots are a common feature of macromolecular recognition. Because, at present, in silico calculations cannot reliably identify hot spots, our quantitative and systematic investigations of an RNA−protein interface may be used to develop better computational approaches.(36)

To cleave the SRL ~1000-fold faster than a single-stranded nucleic acid substrate, ssNA, restrictocin recognizes its substrate not during the first but rather the second kinetic step of the reaction, k2, which involves site-docking and cleavage (Figure (Figure11A).3,4 The importance of SRL motifs to docking was probed by comparing k2 values for restrictocin or its mutants cleaving SRL variants with one or both motifs missing. Removal of the bulged-G motif from the SRL substrate results in a 64-fold loss in activity with an additional 15-fold loss upon complete removal of secondary structure (absence of the bulged-G motif, tetraloop, and stem structure). These findings indicate that the majority of the contribution to specificity arises from contacts to the bulged-G motif, in the context of the SRL, in accord with previous studies.3,17,37 The data in Figure Figure33 indicate that the lysine triad (K110, K111, and K113) and in particular K113 contributes to bulged-G recognition. Our 7dN data and mutational data of others(22) show that each residue in the lysine triad is needed for full activity. Given the juxtaposition of these side chains, it is reasonable to expect that mutating one would affect positioning of the other two (Figure (Figure1D).1D). Even though bulged-G motif recognition contributes to catalysis, recognition relies as well on additional tetraloop contacts to active site residues.

Previous qualitative studies indicated the importance of Y47 and H49 to specificity.19,21 Here, our kinetic analysis provides insight into the molecular basis of the linkage between the mode of substrate docking and catalysis with different substrates engaging active site residues Y47 and H49 to different degrees. Structural and functional studies2,9 provide evidence that the active site histidine (H49) does not make sequence specific contacts to the substrate (Figure (Figure1C).1C). Rather, it contributes to transition state stabilization and is expected to contact the nucleophilic 2′ hydroxyl group (Figure (Figure1C).1C). If all nucleic acid substrates docked similarly, mutation of H49 would have affected the activity of every substrate to a similar extent. Our kinetic studies do not support this model. Restrictocin cleaves the ssNA substrate with no detectable contribution from the active site residues Y47 and H49 (Figure (Figure3B).3B). In contrast, this enzyme cleaves the tetraloop substrate by engaging Y47 but not H49 (Figure (Figure3D)3D) and cleaves the SRL and 7dN substrates with participation of both active site residues (Figure (Figure3C).3C). These substrate-dependent contributions of active site residues to catalysis likely reflect different modes of docking for cognate and noncognate substrates (Tables (Tables11 and and22 and Figures Figures33 and and4).4). Evidently, bulged-G recognition is needed to engage H49, whereas the tetraloop by itself appears sufficient to engage Y47.

The simplest model to account for the Y47F and H49A data involves at least two modes of substrate docking into the active site (Figure (Figure5).5). In the nonspecific mode, docking of non-SRL substrates permits only partial engagement of the catalytic residues and results in an impaired cleavage rate. In contrast, docking in the specific mode with the SRL enables full engagement of the catalytic machinery (including Y47 and H49) to achieve a ~1000-fold rate enhancement of site-cleavage of the SRL relative to cleavage of ssNA substrates.

The molecular basis by which different substrates elicits two docking modes is unknown, but two attractive candidate mechanisms exist. In the first, active site docking of the SRL places its nucleophile and scissile bond in position close enough to engage the side chains of Y47 and H49. For docking of non-SRL substrates, these side chains are farther away from the nucleophile and scissile bond because only the SRL possesses the contacts necessary to stabilize the specific docking mode. Hence, the nonspecific docking mode results in slower cleavage rates. In the second mechanism, only SRL docking triggers an induced fit in the RNA, protein or both to shorten the distance and thereby engage Y47 and H49 catalytically.

The two modes of docking with differential activity create a kinetic discrimination mechanism to ensure that the SRL is cleaved faster than non-SRL substrates. This mechanism of active site docking for the SRL substrate offers functional support for the structural model of site docking (Figure (Figure1C,D1C,D and the upper right panel of Figure Figure5)5) in which both SRL motifs are simultaneously recognized. As a result, the target nucleotide is positioned in a base-flipped form to orient it for maximal activity.

Recognition of RNA by restrictocin exemplifies two recurrent themes in RNA−protein recognition.(38) Groove binding is illustrated by the lysine triad loop recognizing the enlarged major groove of the SRL and its distinctive bulged-G nucleobase. β-Sheet binding is illustrated by the unfolded tetraloop that docks into the active site pocket. Our work shows that restrictocin couples these recognition strategies to achieve specificity.

Acknowledgments

We are grateful to Y.-L. Chan, J. Olvera, and I. G. Wool for valuable advice and discussion and to N. Gao for technical assistance.

Glossary

Abbreviations Used

SRL
sarcin/ricin loop
Wt
wild type
E:S
electrostatic Michaelis complex
tetraloop substrate
SRL in which bulged-G motif is replaced with Watson−Crick stem
7dN substrate
SRL in which the bulged-G contains a 7 deazaguanosine
ssNA substrate
oligonucleotide with a single riboguanosine embedded in single-stranded DNA

Funding Statement

National Institutes of Health, United States

Notes

This work was supported by grants to C.C.C. from the National Institutes of Health (GM59872 and GM070491) and to J.A.P. from Howard Hughes Medical Institute.

Author Present Address

Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine & Science.

Author Present Address

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.

Author Present Address

Department of Laboratory Oncology Research, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, Savannah, GA 31404.

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