Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA in the peptidyl transferase center

K. Mark Parnell*, Amy C. Seila†, and Scott A. Strobel*‡§

Departments of *Molecular Biophysics and Biochemistry, †Genetics, and ‡Chemistry, Yale University, 260 Whitney Avenue, New Haven, CT 06520-8114

Edited by Harry F. Noller, University of California, Santa Cruz, CA, and approved July 16, 2002 (received for review April 8, 2002) The crystal structure of the ribosomal 50S subunit from Haloarcula activity, suggesting that 23S and 5S rRNA may constitute the marismortui in complex with the transition state analog CCdA- bulk of the peptidyl transferase center (8). -puromycin (CCdApPmn) led to a mechanistic proposal The most unambiguous evidence that the active site of the wherein the universally conversed A2451 in the ribosomal active ribosome is comprised of RNA came from the 2.4-Å crystal site acts as an ‘‘oxyanion hole’’ to promote the peptidyl transferase structure of the Haloarcula marismortui 50S ribosomal subunit reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. reported by Ban et al. (9) and Nissen et al. (10). The structure of the (2000) Science 289, 920–929]. In the model, close proximity (3 Å) 50S subunit complexed with the transition-state analog CCdA- between the A2451 N3 and the nonbridging phosphoramidate phosphate-puromycin (CCdApPmn) was vital to this structural of CCdApPmn suggested that the carbonyl oxyanion identification. CCdApPmn includes the minimal components of formed during the tetrahedral transition state is stabilized by both peptidyl transferase substrates (11). CCdA binds the P site, and hydrogen bonding to the protonated A2451 N3, the pKa of which puromycin binds the A site (Fig. 1). The ␣-amino of must be perturbed substantially. We characterize the contribution puromycin is connected covalently to the 3Ј oxygen of CCdA of the putative hydrogen bond between the N3 of A2451 and the through a phosphoramidate linkage that has a tetrahedral geometry nonbridging phosphoramidate oxygen by using chemical protec- comparable in shape to the peptidyl transfer transition state (11, tion and peptidyl transfer inhibition assays. If this putative hydro- 12). Within the cocrystal structure only 23S rRNA contacts gen bond makes a significant thermodynamic contribution, then CCdApPmn. In fact, the nearest protein residue is almost 20 Å CCdApPmn-binding affinity to the 50S ribosomal subunit should be removed from the phosphoramidate linkage, which argues that strongly pH-dependent, with affinity increasing as the pH is low- rRNA and not protein catalyzes peptidyl transfer (10). ered. We report that CCdApPmn binds 50S ribosomes with essen- Within the 50S structure, the N3 of the universally conserved tially equal affinity at all pH values between 5.0 and 8.5. These data A2451 (Escherichia coli numbering) is within hydrogen-bonding argue against a mechanism for peptidyl transfer in which a residue distance (3 Å) of the nonbridging phosphoramidate oxygen with near neutral pKa stabilizes the transition-state oxyanion, at (designated O2 in the crystal coordinates) of CCdApPmn (Fig. least to the extent that CCdApPmn accurately mimics the transition 1a). The implied hydrogen bond occurs despite the fact that state. neither the O2 oxygen nor the N3 imino group would normally be protonated at pH 5.8, the pH used for crystallization of the he ribosome is a molecular machine that assembles polypep- 50S subunit (10). Based on the assumption that O2 is analogous Ttide chains. The addition of an amino acid onto a nascent to the negatively charged oxyanion formed in the transition state, peptide chain, termed peptidyl transfer, is catalyzed by the 50S the close approach of these two groups led Nissen et al. to two ribosomal subunit by using aminoacyl-tRNA and peptidyl-tRNA conclusions: (i) the N3 pKa of A2451 is perturbed toward as substrates. In the course of the reaction, the peptidyl-tRNA, neutrality in the transition state, and (ii) the protonated A2451 charged with the growing peptide chain, occupies the P site, and N3 stabilizes the negative charge on the transition state oxyanion an aminoacyl-tRNA, activated with a single amino acid, binds by hydrogen bonding and͞or charge neutralization. In this the A site. Peptide bond formation occurs by a transacylation manner A2451 was proposed to serve as the oxyanion hole for reaction mechanism wherein the ␣-amino group on the A-site the peptidyl transfer reaction (ref. 10; Fig. 1b). This contribution tRNA nucleophilically attacks the ester linkage between the is in addition to its role as a general base for activation of the peptide chain and the 3Ј-hydroxyl of the P-site tRNA. It is nucleophilic ␣-amino group. expected that the reaction proceeds through a transition state Biochemical experiments have attempted to determine the that has a tetrahedral geometry at the carbonyl carbon and extent to which the A2451 pKa is perturbed in the ground state includes a negatively charged oxyanion. Collapse of the transi- of the ribosome. The pH dependence of dimethyl (DMS) tion state produces a deacylated P-site tRNA and a peptide chain reactivity at A2451 suggested that the active-site residue has an that is elongated by one amino acid coupled to the A-site tRNA unusually high pKa of 7.6 (13). However, subsequent experi- (for review see ref. 1). ments found that DMS modification at A2451 occurred only in Defining how this reaction is catalyzed has been a question of inactive E. coli 50S subunits, and no reactivity was observed at active research for over 30 years. Despite an early and rather any pH after heat activation (14). Furthermore, the DMS indirect indication that a protein side chain might be responsible modification most likely occurred at the N1 rather than the N3 for catalysis (2, 3), biochemical evidence has identified RNA, imino group of A2451 (15). DMS protection experiments per- which accounts for about two thirds of ribosomal molecular formed on ribosomes from several different organisms showed weight (4), as the most likely catalytic component. Highly either no reactivity at A2451 or a reactivity pattern inconsistent conserved internal loops of the 23S rRNA domain V have been with a direct pKa effect (15, 16). Overall, these data are more shown biochemically to interact with the 3Ј-CCA ends of the A-site and P-site tRNAs (5, 6) as well as aminoacyl residues attached to the P-site tRNA (7). These results showed that rRNA This paper was submitted directly (Track II) to the PNAS office. is in close proximity to the nucleophile and leaving group. Other Abbreviations: CCdApPmn, CCdA-phosphate-puromycin; DMS, dimethyl sulfate; pcb, phe- experiments showed that large ribosomal subunit particles nylalanyl-caproyl-biotin; CPmn, C-puromycin. stripped of 95% of the ribosomal protein retained catalytic §To whom reprint requests should be addressed. E-mail: [email protected].

11658–11663 ͉ PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.182210099 Downloaded by guest on October 2, 2021 be higher at acidic pH and become progressively weaker as the pH is raised. We have explored this hypothesis by measuring the pH dependence of CCdApPmn binding. The results argue against transition-state oxyanion stabilization by the peptidyl transferase center of the ribosome insofar as CCdApPmn is an accurate mimic of that transition state. Materials and Methods Synthesis of CCdApPmn. The synthesis of CCdApPmn followed that described by Welch et al. (11) with minor modifications. CCdAp (100 nmol, Dharmacon Research, Lafayette, CO) was coupled to puromycin (12.3 ␮mol) in the presence of 1-ethyl-3- [3-(dimethylamino)propyl]carbodiimide (EDAC, 50 ␮mol) buff- ered with 400 mM Mes at pH 6.0 in an aqueous reaction volume of 125 ␮l. The reaction was carried out for4hat25°C. The mixture then was diluted to 500 ␮l with water and added to a 200-␮l A-25 Sephadex column. The column was washed with 4 ml of water, followed by 4 ml of 30 mM NH4OAc (pH 6.5), and eluted with 4 ml of 750 mM NH4OAc. CCdApPmn was purified further by HPLC with a C18 column (Rainin Instruments) and ͞ eluted with a 100 mM NH4OAc (pH 6.5) acetonitrile gradient (0–50% acetonitrile) over 80 min. The retention time of CCdApPmn was 38 min. Product formation was confirmed by mass spectrometry (Howard Hughes Medical Institute͞Keck Biotechnology Resource Laboratory, Yale University, New Ha- ven, CT) and 31P NMR: theoretical m͞z, 1,393.36; actual m͞z, 1393.35. 31P NMR: ␦, Ϫ0.09, 0.14, 5.79 ppm. BIOCHEMISTRY Preparation of Ribosomes. Ribosomal 50S subunits were isolated from early log-phase MRE600 cells and prepared as described by Rheinberger et al. (17) with minor modifications.

Chemical Modification of 23S rRNA. The 23S rRNA within intact ribosomal particles was modified with DMS. Each reaction contained 50 nM 50S ribosomes (preheated at 42°C for 5 min), ͞ 200 mM KCl, 20 mM MgCl2, 50 mM buffer (KOAc, pH 5.0 Mes, pH 5.5, pH 6.0͞Mops, pH 6.5, pH 7.0, pH 7.5͞Tris-Cl, pH 8.0͞Tris-, pH 8.5), 33% methanol, and 0–1,500 nM CCdApPmn in a total volume of 25 ␮l. DMS was added to ribosomes (1 ␮l of a 1:10 DMS͞ethanol solution prepared Fig. 1. Peptidyl transferase transition-state inhibitor and proposed mecha- immediately before the reaction) and allowed to react at 25°C for nism of transition-state stabilization based on the inhibitor structure. (a) 30 min. The RNA was precipitated by adding 2.5 volumes of Schematic representation of CCdApPmn in the peptidyl transferase center of ethanol and stored at Ϫ80°C for 4 h, after which the ribosomes the ribosome, indicating the stereochemistry about the phosphoramidate. were pelleted by centrifugation. The rRNA was isolated from The reported distance from the N3 of A2451 to the nonbridging O2 of the ribosomal proteins by phenol͞chloroform extraction. achiral phosphoramidate linker of CCdApPmn is indicated (10). (b) Mechanism of transition-state oxyanion stabilization by a protonated N3 of A2451 based Primer Extension and Gel Electrophoresis. The extent of DMS on the proposal of Nissen et al. (10). The chirality of the transition state is modification at A2602 was monitored by reverse transcription indicated, with presumed stereochemistry and assignments of oxyanion and froma5Ј-32P-radiolabeled DNA primer complementary to CH of ␣-carbon of peptide chain based on the crystallographic model (10). The A-site tRNA is depicted as being charged with tyrosine. In both cases, only the nucleotides 2,639–2,656. The DNA primer (50,000 cpm) and 23S chirality of the tetrahedral center is indicated. rRNA (10 nM) were heat-denatured at 90°C for 2 min in annealing buffer (50 mM Tris, pH 8.3͞50 mM NaCl͞10 mM DTT) in a 5-␮l total reaction volume and slow-cooled to 42°C. avian myeloblastosis virus reverse transcriptase (2 units, Roche consistent with a pH-dependent conformational change in the ␮ peptidyl transfer center that involves A2451 (15), but they do not Applied Science, Indianapolis), dNTPs (80 M final concentra- address whether the N3 pKa is perturbed either in the ground or tion each), and MgCl2 (10 mM final concentration) were added, and the reaction was incubated for 30 min at 42°C. Adenosine transition states of the reaction. ladders were obtained by adding a final concentration of 5 ␮M At present, the only evidence to support an elevated pKa for ddUTP to reverse transcription of unmodified 23S rRNA. A2451 in the transition-state complex is the interatomic distance Reactions were quenched by the addition of an equal volume of between the A2451 N3 and the phosphoramidate O2 in the formamide loading buffer (95% formamide͞25 mM EDTA, pH cocrystal structure (10). This distance implies hydrogen-bond 8.0) and heat-denatured at 90°C for 2 min. Experiments at each formation, but for this to occur the base must be protonated at pH were run in duplicate. a pH well above its regular pKa. Thus, the structure makes a Primer extension products were resolved by 8% denaturing biochemical prediction that can be tested experimentally. If the polyacrylamide electrophoresis and quantitated by using a Mo- putative hydrogen bond between A2451 and the phosphorami- lecular Dynamics STORM PhosphorImager. The DMS modifi- date contributes to CCdApPmn-binding affinity, then its binding cation at A2602 results in a reverse-transcriptase stop that is constant should be strongly pH-dependent. The affinity should visible as a band on a polyacrylamide sequencing gel occurring

Parnell et al. PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 ͉ 11659 Downloaded by guest on October 2, 2021 1 nt before the modified position. CCdApPmn binding results in Results reduced DMS accessibility at A2602, which attenuates the band We set out to measure the contribution of the putative hydrogen intensity. The loss in intensity of DMS modification at A2602 (I) bond between the N3 of A2451 and the O2 nonbridging phos- as a function of CCdApPmn concentration was used to deter- phoramidate oxygen as a means to address if the N3 has a mine the dissociation constant (Kd) of the inhibitor at each pH near-neutral pKa in the transition state. At pH values below its by using the equation pKa, the N3 should be protonated and hydrogen-bond to the phosphoramidate O2 of CCdApPmn, resulting in greater bind- ϭ ϩ ͑ Ϫ ͒͑͞ ϩ ͓ ͔͞ ͒ I Isat I0 Isat 1 CCdApPmn Kd , [1] ing affinity. As the pH is increased, the N3 should be protonated less efficiently, and the inhibitor binding affinity should be where Isat is the band intensity at saturating inhibitor, and I0 is reduced, eventually resulting in a minimum binding constant. the band intensity with no inhibitor at a given pH. Band Thus the pKa of N3 in the transition state can be determined by intensities were normalized for loading and overall DMS reac- analyzing Kd values for CCdApPmn as a function of pH. Toward tivity within 23S rRNA, and band intensities with inhibitor this goal, we used both chemical protection and peptidyl transfer Ͼ present ([CCdApPmn] 0) were normalized relative to I0, the inhibition experiments to measure the pH dependence of band intensity with no inhibitor ([CCdApPmn] ϭ 0). CCdApPmn affinity.

Peptidyl Transfer Fragment Assay Inhibition. Inhibition of peptidyl Chemical Protection of A2602. The universally conserved residue transfer by CCdApPmn was monitored by using a modified A2602 is located near the peptidyl transfer center of the ribo- fragment assay that uses CCA-phenylalanyl-caproyl-biotin some. Within the cocrystal structure, A2602 interacts extensively (CCA-pcb) as the P-site substrate and C-puromycin (CPmn) as with the puromycin moiety of CCdApPmn (10). Consistent with the A-site substrate (18). The reaction products are CPmn- this observation, chemical footprinting experiments demon- strated that A2602 shows a decrease in DMS reactivity after phenylalanyl-caproyl-biotin (CPmn-pcb) and CCA, and both binding of A-site tRNA as well as an enhanced DMS reactivity products are produced at equivalent rates, although the rate of after binding of P-site tRNA (19). DMS footprinting studies CCA formation must be corrected for background . showed that A2602 is modestly protected after binding of Reactions contained 50–75 nM 50S ribosomal subunits (pre- CCdApPmn (11), perhaps a convolution of enhancement and heated for 5 min at 42°C), 200 mM KCl, 20 mM MgCl2,50mM protection because the transition-state analog contains both buffer (KOAc, pH 5.0͞Mes, pH 5.5, pH 6.0͞Mops, pH 6.5, pH ͞ ␮ A-site and P-site tRNA elements. Fig. 2a shows the roughly 7.0, pH 7.5 Tris-Cl, pH 8.0), 40–500 M CPmn, 200 nM 2-fold reduction in A2602 DMS modification at a saturating 32 unphosphorylated CCA-pcb, 2,000–3,000 cpm of P-labeled CCdApPmn concentration. Also shown is a modest stimulation CCA-pcb, 33% methanol, and either 0 or 200 nM CCdApPmn of U2555 toward DMS reactivity, which was noted previously ␮ in a total volume of 25 l. Although methanol is not necessary (20). We used the protection of A2602 from DMS modification for the modified fragment assay, it increases the rate at low as a means of determining the Kd of CCdApPmn as a function ribosome and substrate concentrations (K.M.P., unpublished of pH. Fig. 2b shows two examples of Kd curves for the inhibitor data). Reactions were initiated by the addition of 50S subunits measured at pH 5.0 and 8.5, the extremes of the pH range Ϯ and incubated at 25°C. Reaction time points were obtained by measured. The curves correspond to a Kd of 220 50 nM for pH ␮ Ϯ removing 2 l of the reaction and quenching it with an equal 5.0 and a Kd of 220 40 nM for pH 8.5. We also measured volume of formamide loading buffer (95% formamide͞50 mM CCdApPmn Kd values at between pH 5.0 and 8.5 (Fig. 2c) Tris-sodium phosphate, pH 6.5). and found that the CCdApPmn binding affinity is independent Products were resolved by 12% denaturing polyacrylamide of pH within this range, which runs counter to the crystallo- electrophoresis. Gels were run at 4°C by using a low-pH gel graphic expectation (10). ͞ running buffer (50 mM Tris NaHPO3, pH 6.5) to prevent base Previous studies also used the modifying agent 1-cyclohexyl- hydrolysis of CCA-pcb. The fraction reacted was quantitated 3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate with a Molecular Dynamics STORM PhosphorImager. These (CMCT) at U2585 to determine the inhibitor binding constant. experiments monitored the conversion of CCA-pcb to CCA. We measured the binding of CCdApPmn by CMCT footprinting Rates were determined by fitting the fraction reacted as a and found the affinity to be essentially invariant between pH 7.5 function of time to an exponential function and subtracting rates and 9.1 (data not shown). Unfortunately this technique is not amenable to pH values below 7.5 because of nonreactivity of the of background hydrolysis. Ki values were determined by Line- weaver–Burke analysis in which rates of both uninhibited and uridine at lower pH (21). Nevertheless, within the pH range that could be measured, the CMCT data are in agreement with that inhibited reactions were plotted as a function of A-site substrate obtained from DMS modification experiments. (CPmn) concentration by using the formula Inhibition of Peptidyl Transfer. As an independent approach to 1͞k ϭ ͑K ͞k ͒͑1͓͞S͔͒͑1 ϩ ͓I͔͞K ͒ ϩ 1͞k , [2] peptidyl transfer M max I max chemical protection, we also measured the CCdApPmn inhibi- tion constant (Ki) as a function of pH. CCdApPmn acts as an where kpeptidyl transfer is the measured rate, [S] is the A-site inhibitor of peptidyl transfer by competing with both A-site and substrate concentration, KM refers to the affinity of the A-site P-site substrates. Under competitive conditions at pH 8.0, substrate for the ribosome, kmax is the maximum rate, [I]isthe Ϯ CCdApPmn was shown to have a Ki of 90 4 nM, which CCdAPmn concentration, and K is the inhibition constant. Both Ϯ I correlated with the Kd of 70 20 nM measured by chemical KM and kmax were determined by least-squares analysis for the protection under similar conditions (11). ϭ uninhibited ([I] 0) reactions at each pH and then used to We determined the Ki of CCdApPmn as a function of pH by determine KI in the inhibited reactions at that pH. Experiments monitoring inhibition of the modified fragment assay on 50S at each pH value were performed in duplicate. The pH values of subunits (18). Over a range of pH values from 5.0 to 8.0, the rates all buffers were measured at 25°C, the same temperature as the of peptidyl transfer were determined as a function of A-site peptidyl transfer reactions. The efficiency of inhibition was not substrate (CPmn) concentration for both inhibited (200 nM affected by preincubation of CCdApPmn with 50S subunits for CCdApPmn) and uninhibited (no CCdApPmn) modified frag- extended periods (data not shown). ment assays. Fig. 3a shows the inhibitory effect that the addition

11660 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182210099 Parnell et al. Downloaded by guest on October 2, 2021 the CCdApPmn phosphoramidate O2 oxygen at low pH might be counterbalanced by a corresponding decrease in affinity of the CCdA and CPmn portions of the inhibitor. We found that the affinities of both substrates are essentially invariant from pH 5.5 to 8.0 (data not shown), which would seem to exclude this possibility. Thus, the hypothesis tested, namely that the N3 of A2451 hydrogen-bonds with a phosphoramidate oxygen of CCdApPmn in a pH-dependent manner, is not supported by the experimental data. Discussion Role of A2451 N3 as a Possible Oxyanion Hole. These experiments set out to address a specific question with regard to the proposed ribosomal peptidyl transferase reaction mechanism: Does A2451 N3 act as the oxyanion hole to stabilize the transition state? To act in such a manner the pKa of A2451 N3, which normally is highly acidic (pKa Յ 1), would have to be perturbed toward neutrality, and the interaction with the transition state would be highly pH-dependent. In contrast to this hypothesis, we find that the interaction between the ribosome and the transition-state analog, CCdApPmn, is pH-independent. Our results cast doubt on the thermodynamic and mechanistic significance of the proposed hydrogen-bonding interaction between the A2451 N3 and the phosphoramidate O2 modeled in the cocrystal structure. Although our results do not rule out the possibility that the A2451 N3 pKa is perturbed to some degree, they put an upper limit on the magnitude of the perturbation. Assuming that the

trend toward weaker binding is reversed at pH values below 5.0, BIOCHEMISTRY our results argue that the pKa is no higher than ϷpH 4.5. Such a pKa would result in a very small fraction (Ͻ5%) of protonated Fig. 2. pH dependence of CCdApPmn binding based on DMS footprinting. N3 residues within the crystal structure at pH 5.8, which argues (a) Protection from DMS modification at A2602 by CCdApPmn binding at pH 7.0. 23S rRNA was reverse-transcribed with a radiolabeled DNA primer com- that the contact modeled in the crystal structure is not indicative plementary to nucleotides 2,639–2,656. Lane 1, rRNA adenosine ladder gen- of transition-state stabilization. erated by incorporation of ddUTP during reverse transcription; lane 2, reverse In order for the N3 of A2451 to have a near-neutral pKa of transcription of 23S rRNA that was not DMS-modified; lane 3, DMS modifica- 7.3–7.7, as measured for the pKa of the peptidyl transferase tion and reverse transcription in the absence of CCdApPmn [note the strong reaction (2, 3), it would need to be perturbed dramatically. 15N new (ϩ1) reverse-transcription stop that reflects modification of A2602]; lane NMR (22, 23) as well as laser Raman spectroscopy (24, 25) 4, DMS modification in the presence of 500 nM CCdApPmn and reverse studies of adenosine nucleosides and nucleotides showed that N3 transcription [note the reduction of signal intensity at A2602 and additionally is not protonated even at pH 1.0, indicating that its pKa must be ϩ the increase in signal intensity at U2555 ( 1)]. (b) Normalized extent of DMS lower than 1.0. At 37°C, a shift from Ϸ1.0 to 7.3 would reactivity at A2602 as a function of CCdApPmn at pH 5.0 and 8.5. For each plot ⅐ Ϫ1 the band intensity was corrected for loading and overall DMS reactivity within correspond to an energetic cost of as much as 9 kcal mol .Itis not evident from the structure how the ribosome could accom- the 23S rRNA. The Kd value for CCdApPmn in the ribosomal active site was calculated by using Eq. 1.(c) pH dependence of CCdApPmn binding as mea- plish a perturbation of this energetic magnitude. A charge-relay sured by chemical protection. Each point is an average of two independent mechanism involving a solvent-inaccessible phosphate and experiments. Standard deviations are indicated with error bars. G2447 was proposed to be responsible for the A2451 N3 pKa perturbation, but G2447 mutations expected to disrupt such a charge relay have little effect on ribosome activity in vivo or in of CCdApPmn has on peptidyl transfer, and Fig. 3b is a vitro (26). representative example of competitive inhibition by CCdApPmn An alternative hypothesis consistent with the crystal structure at pH 7.0. The inhibition of peptidyl transfer in Fig. 3b corre- is that the phosphate O2 of CCdApPmn, and not A2451 N3, is Ϯ sponds to a Ki value of 170 10 nM. We used peptidyl transfer protonated. Because the pKa of the phosphoramidate oxygen is inhibition to measure binding affinities from pH 5.0 to 8.0 and Ϸ3.1 (27), such an interaction also would have resulted in affinity again found that the Ki values did not change with pH (Fig. 3c). that increased inversely with pH. The binding studies seem to The Ki values are relatively constant (125–175 nM) between pH exclude this possibility as well. The data can be generalized to 6.0 and 8.0. At pH values of 5.5 and 5.0 the Ki values increase argue that no functional group with a near-neutral pKa in the Ϸ 3-fold to 220 and 410 nM, respectively. This change in Ki at low ribosome makes a meaningful thermodynamic contribution to pH is in the direction opposite to that predicted by the model. CCdApPmn binding. The measured Ki values are in reasonable agreement with Kd values measured by DMS protection (see above) as well as the Quality of CCdApPmn as a Mimic of the Ribosome Transition State? Ϯ previous Ki measurement at pH 8.0 (90 4 nM) (11). The CCdApPmn has been used to provide both structural evidence chemical protection (Kd) and peptidyl transfer inhibition (Ki) for and biochemical evidence against the proposal that A2451 studies overlap in pH and together span the range from pH 5.0 N3 acts as an oxyanion hole. This evidence, both pro and con, to 8.5. Within this range we observe no trend toward increased is relevant only as far as CCdApPmn is a good mimic of the CCdApPmn affinity for the ribosome at progressively acidic pH transition state. Although the phosphoramidate geometry of values. CCdApPmn is tetrahedral, and the bond length from phospho- In light of observations of a pH-dependent conformational rus to a nonbridging oxygen (1.5 Å) is nearly the same as the change in the ribosomal active site (15), we considered the bond length from carbon to oxygen (1.4 Å) (28), the charge of possibility that an increased affinity between A2451 N3 and the phosphoramidate is delocalized between the two nonbridg-

Parnell et al. PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 ͉ 11661 Downloaded by guest on October 2, 2021 ing . In contrast, the actual transition state contains a single charged oxyanion. The difference in charge distribution between the analog and the transition state may result in a crystal structure conformation about the phosphoramidate that is some- what different from what occurs during peptidyl transfer. Another important difference between the transition state and its phosphoramidate mimic is the absence of a 2Ј-OH on the terminal adenosine of CCdApPmn. Barta et al. suggested that the terminal dA of the transition-state analog would cause it to be a poor mimic of the peptidyl transfer transition state.¶ In the crystal structure, the deoxyadenosine C2Ј of CCdApPmn is within 2.8 Å of the phosphoramidate O2. Were the 2Ј-OH present on the adenosine as it is in a P-site tRNA, it would cause significant steric clash with the phosphoramidate O2 as the complex is currently modeled. The interactions about the tran- sition state during peptidyl transfer therefore are likely to be different from those modeled in the cocrystal structure. In addition to subtle ways in which CCdApPmn may not mimic the tetrahedral transition state, mechanistic proposals based on its structure must contend also with stereochemical ambiguity. Peptidyl transfer by the ribosome proceeds through a chiral tetrahedral transition state, whereas the phosphoramidate of CCdApPmn is achiral. As such, it was necessary to assign either the phosphoramidate O1 or O2 as the oxyanion, whereas the other was designated as the ␣-carbon of the peptide chain (Fig. 1 a and b). Based on the geometry of the inhibitor in the 50S active site, Nissen et al. (10) assigned the oxyanion to be the O2. This assignment led to the proposed interaction between A2451 N3 and the oxyanion. Some consideration must be given to the possibility that the phosphoramidate O1 instead of O2 may correspond to the oxyanion. In this case, A2451 N3 would interact with the ␣-carbon CH, while the oxyanion would be exposed to solvent (the nearest residue is more than 5 Å away). It would follow from this stereochemical assignment that the ribosome does not explicitly stabilize the oxyanion of the tran- sition state, a model that is consistent with our binding results. Hence, the close distance seen between A2451 N3 and the phosphoramidate O2 may in fact be an energetically unfavorable interaction that is overcome by binding of the analog to both the A site and P site.

Role of A2451 as Possible General Acid or Base. The proposed role of A2451 as an oxyanion hole is part of a greater overall mechanistic proposal wherein A2451 plays a prominent role in peptide bond formation (10). In this model, the N3 of A2451 acts first as a general base to abstract a proton from the nucleophilic ␣-amino group or the protonated ␣- groupʈ of the A site-bound aminoacyl-tRNA. The oxyanion of the tetrahedral transition state then is stabilized by the protonated N3 of A2451. Finally, the A2451 N3 acts as a general acid to transfer its proton to the 3Ј-oxyanion leaving group of the P-site tRNA, resulting in an amide linkage. There has been considerable controversy over the role and importance of A2451. Experiments in which A2451 was mutated to another base showed only modest (2–14-fold) decreases in the rate of peptidyl transfer, implying that A2451 does not play a critical role (26, 29). However, these experiments used recon- Fig. 3. pH dependence of CCdApPmn binding based on inhibition of the stituted ribosomes, which had very slow rates of peptidyl transfer, modified fragment assay. (a) Peptidyl transfer fragment assay inhibition by and chemistry was not shown to be the rate-limiting step. ␮ CCdApPmn at pH 7.0. Reactions contain 62 M CPmn in the presence (200 nM) Therefore, large effects on the faster chemical step by the A2451 or absence of CCdApPmn. Also shown is the extent of background hydrolysis of the radiolabeled substrate CCA-pcb in the absence of CPmn. (b) Lineweav- mutation may have gone unnoticed. Recent experiments, using er–Burke plot of inhibition of the modified fragment assay at pH 7.0 in the a peptidyl transfer assay wherein chemistry is expected to be presence or absence of CCdApPmn inhibitor. The concentrations of CPmn rate-limiting, show a pH dependence of peptide bond formation were 20, 23, 26, 30, 36, 46, 62, 95, or 200 ␮M. The Ki value (125 Ϯ 10 nM) was that indicates that there is an ionizable group within the ribo- calculated by using Eq. 2.(c) The pH dependence of Ki for CCdApPmn. Each point represents an average of two Lineweaver–Burke plots with two sets ¶ of nine CPmn concentrations each. Standard deviations are indicated by Barta, A., Dorner, S. & Polacek, N. (2001) Science 291, 203 (abstr.). error bars. ʈBerg, J. M. & Lorsch, J. R. (2001) Science 291, 203 (abstr.).

11662 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182210099 Parnell et al. Downloaded by guest on October 2, 2021 some with pKa of 7.4 (30). Mutation of A2451 to U results in a corresponds to a ‘‘snapshot’’ at an intermediate stage of peptidyl more than a 100-fold decrease in the rate of peptide bond transfer. Chemical footprinting studies indicate a surprising formation coupled with loss of the ribosome’s ionizable group. amount of pH-dependent conformational flexibility at the active This result argues that A2451 is important for the chemical step site of the ribosome [pKa Ϸ 7.6 (15, 16)]. Although our results of peptide bond formation, possibly through a general base argue that this flexibility does not contribute to stabilization of mechanism, although other explanations involving protonation the transition state, it is possible that interactions and confor- of neighboring residues are possible also (30). mations not seen in the current crystal structure play an impor- The inhibitor binding data presented in this study do not tant role in catalysis. Structural and biochemical experiments address the question of whether A2451 or any other base serves that characterize the ribosome at different stages of peptidyl as a general base in peptide bond formation. However, the transfer will be essential to untangle the peptidyl transfer results argue that the pKa measured for the peptidyl transferase mechanism. reaction does not reflect the pKa of a residue serving as the oxyanion hole. Moreover, these data argue against any residue We thank Yomi Oyelere and Greg Muth for helpful discussions and Lara with a pKa of 5.0 or greater serving as the oxyanion hole, insofar Szewczak and Ashley Eversole for critical reading of the manuscript. This as CCdApPmn is an accurate mimic of the transition state. work was supported by American Cancer Society Research Scholar The co-crystal structure of CCdApPmn with the 50S subunit Grant to Beginning Investigators 02-052 (to S.A.S.).

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