Báo cáo khoa học: Eukaryotic class 1 translation termination factor eRF1 ) the NMR structure and dynamics of the middle domain involved in triggering ribosome-dependent peptidyl-tRNA hydrolysis pptx

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Eukaryotic class 1 translation termination factoreRF1)the NMR structure and dynamics of themiddle domain involved in triggering ribosome-dependentpeptidyl-tRNA hydrolysisElena V. Ivanova1, Peter M. Kolosov1, Berry Birdsall2, Geoff Kelly2, Annalisa Pastore2,Lev L. Kisselev1and Vladimir I. Polshakov31 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia2 Division of Molecular Structure, National Institute for Medical Research, London, UK3 Center for Magnetic Tomography and Spectroscopy, M. V. Lomonosov Moscow State University, RussiaTermination of translation, one of the most complexstages in protein biosynthesis, is regulated by the co-operative action of two interacting polypeptide chainrelease factors, eukaryotic class 1 polypeptide chainrelease factor (eRF1) and eukaryotic class 2 polypep-tide chain release factor 3 (eRF3). The roles of theseKeywordshuman class 1 polypeptide chain releasefactor; NMR structure and dynamics;termination of protein synthesisCorrespondenceV. I. Polshakov, Center for MagneticTomography and Spectroscopy, M. V.Lomonosov Moscow State University,Moscow, 119991, RussiaFax: +7 495 2467805Tel: +7 916 1653926E-mail: vpolsha@mail.ru(Received 15 May 2007, accepted 20 June2007)doi:10.1111/j.1742-4658.2007.05949.xThe eukaryotic class 1 polypeptide chain release factor is a three-domainprotein involved in the termination of translation, the final stage of poly-peptide biosynthesis. In attempts to understand the roles of the mid-dle domain of the eukaryotic class 1 polypeptide chain release factor in thetransduction of the termination signal from the small to the large ribo-somal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMRstructure has been obtained. The overall fold and the structure of theb-strand core of the protein in solution are similar to those found in thecrystal. However, the orientation of the functionally critical GGQ loop andneighboring a-helices has genuine and noticeable differences in solutionand in the crystal. Backbone amide protons of most of the residues in theGGQ loop undergo fast exchange with water. However, in the AGQmutant, where functional activity is abolished, a significant reduction in theexchange rate of the amide protons has been observed without a noticeablechange in the loop conformation, providing evidence for the GGQ loopinteraction with water molecule(s) that may serve as a substrate for thehydrolytic cleavage of the peptidyl-tRNA in the ribosome. The proteinbackbone dynamics, studied using15N relaxation experiments, showed thatthe GGQ loop is the most flexible part of the middle domain. The confor-mational flexibility of the GGQ and 215–223 loops, which are situated atopposite ends of the longest a-helix, could be a determinant of the func-tional activity of the eukaryotic class 1 polypeptide chain release factor,with that helix acting as the trigger to transmit the signals from one loopto the other.AbbreviationsaRF1s, archaeal RFs; eRF1, eukaryotic class 1 polypeptide chain release factor; eRF3, eukaryotic class 2 polypeptide chain release factor 3;HNCA, three-dimensional experiment correlating amide HN and Ca signals; HSQC, heteronuclear single quantum coherence spectroscopy;M-domain, eRF1 middle domain (or domain 2); PTC, peptidyl transferase center of the ribosome; R1, longitudinal or spin–lattice relaxationrate; R2, transverse or spin–spin relaxation rate; Rex, conformational exchange contribution to R2; RF, polypeptide chain release factor(s);S2, order parameter reflecting the amplitude of ps–ns bond vector dynamics; se, effective internal correlation time; sm, overall rotationalcorrelation time.FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS 4223termination factors have been validated in vitro in acompletely reconstituted eukaryotic protein synthesissystem [1]. The two major functions of eRF1 are:(a) recognition of one of the three stop codons, UAA,UAG or UGA, in the decoding center of the smallribosomal subunit; and (b) participation in the subse-quent hydrolysis of the ester bond in peptidyl-tRNA.eRF3 is a ribosome- and eRF1-dependent GTPase thatis encoded by an essential gene, and its role in transla-tion termination requires further elucidation [2].The human eRF1 structure, in the crystal [3] and insolution [4], consists of three domains. The N-termi-nal domain is implicated in stop codon recognition[5–14]. The role of the middle (M) domain will bedescribed in detail below. The C domain of eRF1interacts with the C domain of eRF3 [15–18], and thebinding of both factors is essential for fast kinetics ofthe termination of translation [1]. However, in a sim-plified in vitro assay for measuring polypeptide chainrelease factor (RF) activity, eRF1 deprived of theC domain still retains its RF activity [19].The most characteristic feature of the M domain isthe presence of the strictly conserved GGQ motif[20]. In prokaryotes, there are two polypeptiderelease factors called RF1 and RF2, which are func-tionally equivalent to eRF1 in eukaryotes [21,22]. Inthe Escherichia coli ribosome, the GGQ motif ofRF1 or RF2 is located at the peptidyl transferasecenter (PTC) on the large ribosomal subunit, asrevealed by cryo-electron microscopy [23,24], crystalstructure data [25], and biochemical data [26]. It wassuggested [26] and shown by cryo-electron micros-copy [23,24] and X-ray diffraction [25] that RF2undergoes gross conformational changes upon bind-ing to the ribosome that could possibly allow theloop containing the GGQ motif to reach the PTC ofthe ribosome and to promote peptidyl-tRNA hydro-lysis. A significant conformational change was alsosuggested for eRF1 [27] and demonstrated by mole-cular modeling [28]. It has been suggested that theGGQ motif, being universal for all class 1 RFs andcritically important for functional activity of bothprokaryotic and eukaryotic class 1 RFs, should beinvolved in triggering peptidyl-tRNA hydrolysis atthe PTC of the large ribosomal subunit [20]. Thethree-domain structure of eRF1, with the shape ofthe protein resembling the letter ‘Y’, partly mimicsthe ‘L’-shape of the tRNA molecule, and the Mdomain of eRF1 is equivalent to the acceptor stemof a tRNA [29]. It has also been suggested that theGGQ motif is functionally equivalent to the universal3¢-CCA end of all tRNAs [20]. The evidence in sup-port of this hypothesis is growing [25].Mutations of either Gly in the GGQ triplet wereshown to abolish the peptidyl-tRNA hydrolysis activityof human eRF1 in vitro [20,30], of yeast eRF1 in vivo[3], and of Es. coli RF2 both in vivo and in vitro[31,32]. For instance, GAQ mutants of both RF1 andRF2 are four to five orders of magnitude less efficientin the termination reaction than their wild-type coun-terparts, although their ability to bind to the ribosomeis fully retained upon mutation [31]. Thus, the toxicityof these mutants for Es. coli in vivo can be explainedby their competitive inhibition at the ribosome-bindingsite [32].Together, the M and C domains of human eRF1, inthe absence of the N domain, are able to bind to themammalian ribosome and induce GTPase activity ofeRF3 in the presence of GTP [33].The previously determined relatively low-resolutioncrystal structure [3] (2.7 A˚highest resolution) of theM domain was unable to provide all the necessarydetails of the molecular mechanism of the terminationof translation in the ribosomal PTC. It still remainsunclear how a stop signal can be transmitted from thesmall to the large ribosomal subunit, and how theM domain participates in hydrolysis of the peptidyl-tRNA ester bond. The aim of this work was to deter-mine the structure and obtain dynamic information onthe M domain of human eRF1 in solution, which mayhelp to clarify these important unanswered questions.ResultsResonance assignment1H,13C and15N chemical shift assignments were madefor essentially all the observed protein backbone amideresonances. More than 95% of all observed side-chain1H,13C and15N chemical shifts were also determined.However, at 298 K, backbone signals from residues177–187, the loop containing the GGQ motif, couldnot be detected. For example, no amide signals attrib-utable to G181, G183 and G184 were observed in therelatively empty Gly region of the15N,1H-heteronuclearsingle quantum coherence spectroscopy (HSQC) spec-trum at this temperature. At lower temperatures(278 K), these amide signals can be detected in the15N-HSQC spectra (Fig. 1A), and the assignmentswere confirmed by three-dimensional experimentscorrelating amide HN and Ca signals (HNCA) and15N-NOESY-HSQC experiments. The absence ofamide signals at 298 K appears to be due to fastexchange of these amide protons with water. An alter-native mechanism of line broadening could be relatedto conformational exchange in the GGQ loop, e.g. theNMR structure and dynamics of eRF1 middle domain E. V. Ivanova et al.4224 FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBScis ⁄ trans interconversion within the Gly residues [34].However, in this case, one can expect to detect similarbehavior of the signals from labile and nonlabile pro-tons. A series of13C-HSQC spectra recorded in thetemperature range between 5 °C and 30 °C showedthat the line widths of the Ha signals of the Gly resi-dues named above do not change very much. Thesefacts unambiguously confirm fast exchange of thebackbone amide protons in the GGQ loop with waterat 298 K. Unlike the backbone amide signals, the side-chain signals of Q185 were observed at 298 K andassigned as the only remaining unassigned pair ofH2N.At 278 K, residues Gly181, Gly183 and Gly184are observed in the15N-HSQC spectrum, and eachappears as a group of signals with different intensitiesand slightly different chemical shifts (Fig. 1A), indicat-ing that this part of the GGQ loop exists as a mixtureof several conformational states similar to that foundfor some other proteins [35,36]. The exchange betweenthese conformational states happens at a relativelyslow rate (slower than  1s)1as estimated from lineshape analysis). These small peaks cannot be assignedto the breakdown protein species, because in that casemany other peaks in the protein spectrum should havesimilar minor satellites. Additionally, for several suchpeaks, sequential and intraresidue correlations werefound in the HNCA and1H,15N-NOESY-HSQC spec-tra, confirming the assignment of these satellite peaksto residues G181, G183 and G184. The existence of aABFig. 1.1H,15N-HSQC spectra of the Mdomain of human eRF1. The numbering ofthe residues corresponds to that of the fulleRF1 protein. (A) The Gly region of the1H,15N-HSQC spectrum of the M domain ofhuman eRF1 recorded at 278 K. (B) Thesuperposition of the1H,15N-HSQC spectraof wild-type (red) and G183A mutant (blue)of the M domain of human eRF1 recordedat 298 K. Clearly visible in blue are theresidues that are absent in the spectrum ofthe wild-type protein due to fast exchangewith water.E. V. Ivanova et al. NMR structure and dynamics of eRF1 middle domainFEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS 4225protein fragment in multiple conformational statesreflects the very complex dynamic behavior of theGGQ loop.Effect of G183A mutationA comparison of the spectra recorded at 298 K for thewild-type M domain of human eRF1 and the G183Amutant (where the first Gly residue in the GGQ motifis replaced by Ala) shows that the chemical shifts ofthe vast majority of HN resonances are virtually iden-tical in these two species (Fig. 1B). There are, however,several important differences. In the15N-HSQC spec-trum of the G183A mutant, as well as the new signalfrom the backbone amide of Ala183 (the mutationpoint), one now can also observe signals from theneighboring residues His182, Gly184 and Gly181,which were all absent in the15N-HSQC spectrum ofthe wild-type protein recorded at 298 K. Interestingly,the chemical shifts of these resonances in the G183Amutant are very similar to those detected at lower tem-perature in the wild-type protein, indicating that themutation has little (if any) effect on the conformationof the GGQ loop. At the same time, however, theG183A mutation results in a decrease in the rate ofexchange of the backbone amide protons with water,and the NMR signals from the mutant loop residuesare visible at higher temperature (298 K). Surprisingly,two other signals (Gly216 and Asn262) that wereabsent in the15N-HSQC spectrum of the wild-typeM domain of eRF1 recorded at 298 K are now visiblein the spectrum of the G183A mutant.Structure determinationA family of 25 NMR structures was determined on thebasis of 2338 experimental restraints measured at278 K and 298 K (Tables 1–3). This work made useof standard double-resonance and triple-resonanceNMR methods applied to unlabeled,15N-labeled and15N ⁄13C-labeled samples of the M domain of eRF1.For most of the protein residues, the number of NOEsper residue is between 20 and 40; however, this num-ber is significantly lower for residues 178–184, whichare near the GGQ motif, and for several other loopregion residues.The statistics of the final ensemble are given inTables 1–3, and the superposition of the final calcu-lated family is presented in Fig. 2A (backbone atomsof the M domain of the human eRF1 crystal structure[3] are also shown in red for comparison). The NMRstructures had the lowest target-function values, nodistance restraint violations greater than 0.2 A˚, and nodihedral angle violations > 10°. The representativestructure (first model in the family of 25 NMR struc-tures) was selected from the calculated family, as thestructure closest to the average structure and givingthe lowest sum of pairwise rmsd values for the remain-der of the structures in the family. The rmsd of thecalculated family from the representative structure isTable 1. Restraints used in the structure calculation of the Mdomain of human eRF1.Total NOEs 1975Long range (|i–j| > 4) 428Medium (1 < |i–j| £ 4) 236Sequential (|i–j| ¼ 1) 448Intraresidue 863H-bonds 12Total dihedral angles 214Phi (/)96Psi (w)97Chi1 (v1) 21Residual dipolar couplingsN–H 120Ca–Ha5Table 2. Restraint violations and structural statistics for the calcu-lated structures of the M domain of human eRF1 (for 25 struc-tures). No NOE or dihedral angle violations are above 0.2 A˚and10°, respectively.Average rmsd <S>aSrepFrom experimental restraintsDistance (A˚) 0.020 ± 0.001 0.020Dihedral (°) 4.369 ± 0.204 4.397Residual dipolar coupling (Hz) 0.028 ± 0.002 0.030From idealized covalent geometryBonds (A˚) 0.008 ± 0.0002 0.008Angles (°) 1.377 ± 0.027 1.335Impropers (°) 1.903 ± 0.055 1.867% of residues in most favorableregion of Ramachandran plot89.9 89.9% of residues in disallowed regionof Ramachandran plot0.0 0.0a<S> is the ensemble of 25 final structures; Srepis the representa-tive structure, selected from the final family on the criterion of hav-ing the lowest sum of pairwise rmsd for the remaining structuresin the family.Table 3. Superimposition on the representative structure (Table 2).Backbone (C, Ca, N) rmsd of residues 142–275 0.87 ± 0.36All heavy-atom rmsd of residues 142–275 1.14 ± 0.26Backbone (C, Ca, N) rmsd of the proteinwithout unstructured loop residues 178–1860.70 ± 0.34Backbone (C, Ca, N) rmsd of the core regionof protein (residues 142–174, 200–275)0.38 ± 0.07NMR structure and dynamics of eRF1 middle domain E. V. Ivanova et al.4226 FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBSbelow 0.9 A˚for the backbone heavy atoms. However,most of this value originated from the large contribu-tion from the poorly structured GGQ loop. Excludingthese residues, 175–189, the rmsd for heavy atoms ofthe protein backbone is less than 0.4 A˚. In the Rama-chandran plot analysis, 89.9% of the residues in thewhole NMR family were found in the most favoredregions and none in the disallowed regions.Structure analysisThe conformations of the backbone and side-chains ofthe M domain of human eRF1 are well defined exceptfor the residues (175–189) in the GGQ loop. The back-bone conformation of this loop is discussed below inthe section ‘Geometry of the GGQ loop’.The topology of the M domain of human eRF1 canbe described as a b-core constructed of a sheet formedfrom five b-strands (both parallel and antiparallel),surrounded by four helices, a1–a4 (Fig. 2B). Strand b3has a substantial twist at residues 168–169. The longesta-helix (a1) starts at the end of the GGQ loop and hasa bend at residues 195–196. There are also severalloops of various lengths, the longest of which is theGGQ loop. Another loop of interest starts at theC-terminus of helix a1 and connects with b-strand b4,and has a conformation similar to two short antiparal-lel b-strands with a turn at residue Gly216.The solution structure of the M domain of humaneRF1 presented in this work shows considerable simi-larity to the crystal structure of the M domain of thesame protein [3], but it is far from identical (Fig. 2A).The rmsd of the superposition of the heavy backboneatoms (Ca, N, O and C) of the family of 25 NMRstructures onto the crystal structure for the wholeM domain (residues 140–275) is 3.8 ± 0.2 A˚. An anal-ogous rmsd value for the superposition of the morestructured part of the protein (residues 144–174 and200–272) is much lower, 2.7 ± 0.1 A˚. The relativelylarge value originates mainly from the differences inorientation of the loops and helices, as discussed later.ABCFig. 2. The solution structures of the M domain of human eRF1.(A) The stereo view of the ensemble of the final 25 calculatedstructures superimposed on heavy backbone atoms (Ca, N and C).The poorly structured GGQ loop region (residues 175–189) wasexcluded from the superposition. The crystal structure of theM domain of the human eRF1 [3] is superimposed on the same setof atoms in the representative solution structure and is shown inred. (B) The topology of the M domain of human eRF1 and thesecondary structure elements displayed usingMOLMOL [65]. (C)Representative structure of the GGQ loop of the M domain ofhuman eRF1.E. V. Ivanova et al. NMR structure and dynamics of eRF1 middle domainFEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS 4227Geometry of the GGQ loopThe GGQ loop is the most disordered part of theprotein structure (Fig. 2A). However, this loop con-tains the most important functional motif and shouldtherefore be characterized in detail. The selection of arepresentative conformation for the GGQ loop (resi-dues 177–188) was derived from an analysis of all theconformations found in the family of calculatedNMR structures (Table 4). This was done by deter-mining a representative value for each backbone tor-sion angle (/ and w) and each side-chain torsionangle v1. In many cases, these representative valueswere close to the mean value of the torsion angle inthe family. In other cases, when two or several clus-ters of torsion angle values were observed, the valuefrom the most populated cluster was taken as therepresentative value. These values were then used tobuild up a model of the 177–188 loop (Fig. 2C).There are no interatomic clashes in this model. Thermsd value for the superposition of the heavy back-bone atoms (Ca, C, N and O) of this model onthe corresponding part of the family of calculatedNMR solution structures is 1.32 ± 0.35 A˚. The rmsddecreases to 1.01 ± 0.16 A˚when it is superimposedon 13 selected structures from the family of 25 NMRstructures. The rmsd is similar, 1.02 A˚, for the super-position on the representative structure of the family,and it has a minimum value, 0.76 A˚, for one memberof the NMR family.Backbone dynamicsFigure 3 presents the experimentally obtained relaxa-tion rates R1(longitudinal or spin–lattice relaxationrate) and R2(transverse or spin–spin relaxation rate)and NOE values for the amide15N nuclei measuredat 278 K, and the calculated values of the orderparameter S2reflecting the amplitude of ps–ns bondvector dynamics. The relaxation parameters wereobtained using the model with an axially symmetricTable 4. The geometry of the GGQ loop in the family of 25 NMRstructures of the M domain of human eRF1.ResidueRanges of torsion angles inwhole familyaTorsion angles inrepresentativestructure/wv1/wv1Pro177 )19 ± 3 161 ± 6 )48 ± 2 )20 160 )48Lys178 )72 ± 14 )40 ± 11 )90 ± 21 )64 )43 )60Lys179 )77 ± 13 128 ± 12 )63 ± 30 )70 130 )60His180 )128 ± 17 48 ± 68 )128 ± 93 )120 45 180Gly181 80 ± 51 )4 ± 13 90 0Arg182 )53 ± 58 )22 ± 46 )62 ± 105 )63 )40 )60Gly183 )66 ± 104 )135 ± 73 )87 )170Gly184 )53 ± 44 )23 ± 16 )63 )35Gln185 )90 ± 23 135 ± 7 )110 ± 17 )75 135 )60Ser186 )68 ± 5 148 ± 4 0 ± 110 )73 150bAla187 )64 ± 1 )41 ± 2 )64 )42Leu188 )64 ± 1 )42 ± 1 )110 ± 23 )64 )42baThe mean value in the family of 25 structures and the SD.bThereis no preferred conformation of the side-chain in the family.Fig. 3. The relaxation parameters of the amide15N spin of eachresidue measured at 18.7 T (800 MHz proton resonance frequency)and 278 K. (A) The longitudinal relaxation rate, R1. (B) The trans-verse relaxation rate, R2. (C) The heteronuclear15N,1H-steady-stateNOE value. (D) The order parameter, S2, determined by model-freeanalysis with an assumption of axially symmetric anisotropic rota-tional diffusion. (E) The chemical exchange rate Rex.NMR structure and dynamics of eRF1 middle domain E. V. Ivanova et al.4228 FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBSdiffusion tensor. The order parameter is smallest (thatis, for the most typical types of internal motions, theamplitude of such motions is largest) for residues176–187 and also the N-terminal residues. The chemi-cal exchange contribution to the transverse relaxationrate Rex(conformational exchange contribution to R2)is also shown in Fig. 3. The relaxation parameterswere obtained using the model with an axially sym-metric diffusion tensor. The average correlation time[1 ⁄ (2Dk+4D^] was 20.8 ± 0.8 ns, and the ratio ofthe principal axis of the tensor (Dk⁄ D^) was1.8 ± 0.1. It is necessary to note that the model thatallows the most successful fit of the experimental datais based on two internal motions that are faster thanthe overall rotational tumbling [37]. Figure 4 illus-trates the convergence of the simulated data (redspots) with most of the experimental data (black cir-cles). The synthetic data were calculated assuming theexistence of relatively slow internal motions, occurringwith a 1.1 ± 0.1 ns correlation time and an orderparameter between 0.5 and 1.0, against a backgroundof faster motions occurring with a correlation timebelow 20 ns and an order parameter between 0.8 and1.0. This was calculated without the assumption ofconformational line broadening. The residues thatexhibit slow conformational rearrangements occurringon a millisecond time scale and leading to an increasein the transverse relaxation rate can be found in aregion outside and to the top of the synthetic dataset(Fig. 4). The most atypical residues in this group areD217, I256 and V210. Residues on the right side ofthis plot (i.e. with the largest NOE values) mostlycome from the rigid protein core. Figure 4 provides aclear and useful illustration of the dynamic behaviorof the protein.Figure 5 shows a ribbon representation of theM domain with the cylindrical radius proportional tothe order parameters S2(A) and Rex(B). Interest-ingly, ignoring the trivial case of the N-terminal resi-dues, the two most flexible loop regions in theM domain are situated on the two opposite sides ofthe long helix, a1 (Figs 2B and 5). The GGQ loopexhibits motions occurring with a  1 ns correlationtime, whereas the loop composed of residues 215–223undergoes motions on both the nanosecond and milli-second time scales. Another flexible part of the pro-tein that undergoes motions on both the fast andslow time scales (indicative residue I256) is the begin-ning of the helix a4, which connects to the C domainof human eRF1.DiscussionThe family of class 1 release factorsThe alignment of the amino acid sequences of theM domains of eRF1s and aRF1s (archaeal RFs) fromdiverse organisms, including the evolutionarily distanteRF1s from lower eukaryotic organisms with variantgenetic codes, such as Stylonichia and Euplotes,isshown in Fig. 6. The sequences between Leu176 andAla210 (human eRF1 numbering) are highly conservedand contain, apart from the invariant GGQ motif,some other residues near this motif that are also com-pletely conserved among all species, including membersof the archaea, namely Pro177, Lys179 and Ser186 inthe loop region, and Arg189, Phe190 and Leu193 atthe beginning of the a1 helix. The highly conservedGly residues in positions 163, 183, 184 and 228 mostlikely have a topology-forming role, allowing the pro-tein backbone to have a specific geometry. Severalother highly conserved residues may have a functionalrole by forming an interface for protein–RNA binding.Fig. 4. The distribution of the experimental (black dots) and simu-lated (small red squares) ratios of relaxation rates R2⁄ R1vs. theheteronuclear15N,1H-NOE values. The data were simulated at800 MHz proton resonance frequency using Clore’s extension ofthe Lipari and Szabo model [37]. The axial symmetry with theratio Dk⁄ D^of the principal axis of the tensor was 1.8 ± 0.1; thevalue of effective overall correlation time 1 ⁄ (2Dk+4D^) was20.8 ± 0.8 ns; the values of the order parameter S2slowwerebetween 0.5 and 1.0; the values of the order parameter S2fastwerebetween 0.8 and 1.0; the values of the internal motion correlationtimes sslowwere between 1 and 1.1 ns; and the values of theinternal motion correlation times sfastwere between 0 and 20 ps.E. V. Ivanova et al. NMR structure and dynamics of eRF1 middle domainFEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS 4229The high level of the alignment similarity suggests thatthe tertiary structure of the M domain is well con-served in both eukaryotic and archaeal RFs.The high degree of conservation of the GGQ-con-taining fragment of the M domain is most likely to beassociated with its role in triggering peptidyl-tRNAhydrolysis. As the ribosomal PTC is mostly composedof rRNA, which in turn is also highly conserved acrossspecies [38–40], the conservation of the GGQ-contain-ing fragment is likely to be associated with its bindingto the conserved RNA sequences.Comparison with the crystal structureof human eRF1The most noticeable difference between the crystalstructure of the M domain in the whole protein andthe solution structure of the separated individualABFig. 5. Ribbon representation of the back-bone of the M domain of human eRF1. Thevariable radius of the cylinder is proportionalto the dynamic properties of the protein res-idues. (A) Fast motions (on a picosecond tonanosecond time scale). The thickness ofthe backbone ribbon is proportional to thevalue of 1 ) S2); the minimal thicknesscorresponds to the value S2¼ 1, and themaximum to S2¼ 0.5. (B) Slow conforma-tional rearrangements (occurring on amillisecond time scale). The thickness of thebackbone ribbon is proportional to the valueof Rex; the minimal thickness correspondsto the value Rex¼ 0, and the maximum toRex¼ 10.Fig. 6. Sequences of the M domains ofeRF1 ⁄ aRF1 from Homo sapiens (1), Saccha-romyces cerevisae (2), Schizosaccharomy-ces pombe (3), Paramecium tetraurelia (4),Oxytricha trifallax (5), Euplotes aedicula-tus (6), Blepharisma americanum (7), Tetra-hymena thermophila (8), Stylonychiamytilus (9), Dictyostelium discoideum (10),Archaeoglobus fulgidus (11), Pyrococcusabyssi (12) and Methanococcus janna-schii (13), as aligned using BLAST [71], withminor manual corrections. Highly and com-pletely conserved residues of RFs are indi-cated by dark and light gray, respectively.Identified secondary structure elements inthe M domain of human eRF1 are shownabove the sequence. The numbering abovethe sequence corresponds to human eRF1.NMR structure and dynamics of eRF1 middle domain E. V. Ivanova et al.4230 FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBSM domain as seen in Fig. 2A is the orientation of theGGQ loop and its connection to helix a1. Our confi-dence in the accuracy of the determination of the ori-entation of the flexible GGQ loop in solution is basedon the extensive use of residual dipolar couplingrestraints, both1D(15N,1H) and1D(13C,1H), that showgood agreement between experimental and calculatedvalues of these parameters. There are three possiblereasons for the differences between the crystal and thesolution structures of the M domain. First, the orienta-tion of the loop may change, due to crystal-packingeffects. Second, the coordinates of the GGQ loop maynot be determined by the X-ray data sufficiently well,because of the relatively low resolution and the flexibil-ity of the GGQ loop. It is of note that about 2.8% ofthe eRF1 residues in the crystal structure were foundin disallowed regions of the Ramachandran plot [3],which indicates that experimental problems may haveresulted in a decrease in the overall quality of thestructure. Finally, the C and N domains may havestructural influences on the M domain within thewhole eRF1 protein.The pairwise comparison of the solution structureswith the X-ray crystal structure of the M domain usingthe superposition of five-residue fragments (Fig. 7)shows that the local geometry of regions 177–184,194–195, 213–219, 237–245 and 258–260 is different.All these regions, except 194–197, correspond to loopsthat connect regular secondary structure elements. Res-idues 194–197 are situated at the bend in helix a1, andare not observed in the crystal structure of humaneRF1 [3]. Therefore, the differences between the crystaland solution structures arise mainly from changes inthe orientations of the loops and a-helices relative tothe b-core.Effect of mutationsThe mutation of either Gly residue in the GGQ motifof class 1 RFs has been shown to abolish the RFactivity both in vivo and in vitro. The G183A mutantof human eRF1 was totally inactive in peptidyl-tRNAhydrolysis [20], and it has been proposed that thismutation alters the structure of the GGQ loop [1].However, the replacement of Gly183 by an Ala hasonly minor effects on the chemical shifts of signalsfrom the vast majority of the residues of the M domain(Fig. 1B). This is strong evidence that there is nosubstantial change in the conformation of the proteinor in the distribution of the conformational ensembleof the GGQ loop. In contrast to this lack of effect onthe conformation, the G183A mutation has a drasticeffect on the exchange of amide protons with water.Fast exchange with water of GGQ loop amideprotonsIt was noted above that many of the residues in theGGQ loop were not detected in the NMR spectra ofthe wild-type M domain at room temperature, due tofast exchange with water. Such fast exchange of theamide proton with water can be caused by several pos-sible mechanisms. These include: (a) coordination of awater molecule(s) involved in subsequent exchangewith amide proton, facilitated by appropriate orienta-tion of HN bonds relative to the CO bond [41]; and(b) the local pH being above 8 and thereby allowingthe HNs to exchange rapidly via base catalysis [42].The GGQ loop region has a predominant positivecharge, and this may have implications for the possiblebinding of the protein to rRNA [3]. One of theFig. 7. A plot of the calculated rmsd for the displacements over the backbone atoms (Ca, C and N) calculated from the pairwise superimpo-sition of five-residue segments of the crystal structure on the equivalent segments of each member of the family of the solution structureof the M domain of human eRF1. The resulting rmsd values (y-axis) and their deviations through the 25 NMR structures are shown for thecentral residue of the five-residue segments (x-axis).E. V. Ivanova et al. NMR structure and dynamics of eRF1 middle domainFEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS 4231possible consequences of this charge imbalance couldbe an increase in the local pH. However, the fact thatthe G183A mutation significantly decreases theexchange rate of the amide protons in the loop regionindicates that a higher local pH is unlikely to be thereason for the fast exchange, as the replacement of oneneutral residue by another without a conformationalchange cannot substantially influence the distributionof the local potential. Therefore, most probably, theobserved effect relates to the coordination of a watermolecule(s) in the GGQ loop and its involvement incatalysis of amide proton exchange.The possible water coordination to the GGQ loopmay facilitate an understanding of the mechanism ofpeptidyl-tRNA hydrolysis. It has been suggested thatthe glutamine side-chain in the GGQ minidomain actsto coordinate the substrate water molecule that per-forms the nucleophilic attack on the peptidyl-tRNAester bond and that the conserved adjacent Gly andneighboring basic residues facilitate contact with thephosphate backbone of either rRNA and ⁄ or the accep-tor stem of the P site tRNA [3]. Although this hypoth-esis has not been supported by any experimental data[30,43–45], one can propose, on the basis of the cur-rent observations, that the protein backbone of theGGQ loop could be responsible for the water moleculecoordination.Dynamic properties of the M domainThe dynamic behavior of the M domain has severalimportant features. First of all, the most flexible regionis the GGQ loop, which is also the most importantfunctionally. It undergoes not only very fast (picosec-ond to nanosecond time scale) but also relatively slowconformational rearrangements, occurring on a milli-second to second (and possibly slower) time scale.High mobility is a characteristic of many RNA- andDNA-binding proteins [46–48], and may facilitate eas-ier positional rearrangement of the protein during thedocking to the binding site on the ribosome or otherligands. Strikingly, the second most flexible part of theprotein (if one does not take into account the N-termi-nal region of the M domain) is the loop situated onthe other end of helix a1 from the GGQ motif(Fig. 5). This loop (residues 215–223) undergoes bothfast (with a correlation time of about 1 ns) and slow(millisecond time scale) motions. There are two possi-ble functional implications of the behavior of thisloop. The first is the facilitation of the conformationalrearrangements and the maintenance of the conforma-tional plasticity for effective binding of the protein tothe ribosome. The second, and more plausible, is thatthe loop is situated at the interface between the M andN domains of eRF1, and this flexibility may beinvolved in transduction of the signal from the N-ter-minal domain, upon the recognition of the stop codon,to the M domain for subsequent initiation of thehydrolysis of peptidyl-tRNA ester bond. Two possiblemodels of signal transduction may be considered. Thefirst model assumes that the signal is transmitteddirectly through the body of eRF1 from the N domainto the GGQ loop of the M domain located in thePTC. The second model postulates that rRNA(s) couldmediate the signal transduction through the follow-ing schematic chain: N domain fi 18S rRNA fi 28SrRNA fi M domain fi GGQ fi PTC-peptidyl-tRNA.No evidence is available at present that favors eithermodel; however, the flexibility of the M domain maybe implicated in both models. The long and relativelydynamically rigid helix a1 could serve as a trigger thatfacilitates the conformational change in one loop con-sequent to a change at the other loop.Interestingly, the short loop at the interface betweenstrand b6 and the C-terminal helix a3 also exhibits thetwo types of motion ) slow conformational rearrange-ment occurring on a millisecond time scale, and rela-tively fast motions (with  1 ns correlation time). Thisslow motion was detected from the large increase ofthe transverse relaxation rate of residue I256, occurringat the same time as the fast motions. Helix a3 connectsthe M domain with the C domain of eRF1, and themotions of this short loop could be a reflection of theabsence of the interacting C domain in this construct.Experimental proceduresSample preparationTo construct the pET-MeRF1 vector for expression of thehuman eRF1 fragment encoding the M domain with theC-terminal His6-tag fusion, a PCR fragment derived frompERF4B [6] was inserted between the NdeI and XhoI sites ofpET23b (Novagen, Madison, WI, USA). The M domain(residues 142–275 of human eRF1) was overproduced inEs. coli strain BL21(DE3) in M9 minimal medium. For13Cand ⁄ or15N labeling [13C6]d-glucose and ⁄ or15NH4Cl (Cam-bridge Isotope Laboratories Inc., Andover, MA, USA) wereused as a sole carbon and ⁄ or nitrogen source in M9 minimalmedium. The His6-tagged M domain of human eRF1 wasisolated and purified using affinity chromatography onNi2+–nitrilotriacetic acid agarose (Qiagen, Germantown,MD, USA). Peak fractions were dialyzed against 20 mmpotassium phosphate buffer (pH 6.9) and 50 mm NaCl,and then purified by cation exchange chromatographyusing HiTrap SP columns (Amersham Pharmacia Biotech,NMR structure and dynamics of eRF1 middle domain E. V. Ivanova et al.4232 FEBS Journal 274 (2007) 4223–4237 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... for the protein backbone) of all 25 conformers of the NMR family of solution structures of the M domain of human eRF1 Fig S3 A surface representation of the M domain of human eRF1, mapping the electrostatic potential Fig S4 A comparison of part of the protein backbone structure of the representative solution structure of the human eRF1 M domain and the Ca trace in the crystal structure of RF1 in the. .. Ebihara K & Nakamura Y (19 9 9) C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G -domain uncoupled binding and the role of conserved amino acids RNA 5, 739–750 Merkulova TI, Frolova LY, Lazar M, Camonis J & Kisselev LL (19 9 9) C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction FEBS Lett 443, 41 47 Eurwilaichitr... Soc 10 4, 4559–4570 68 Clore GM, Driscoll PC, Wingfield PT & Gronenborn AM (19 9 0) Analysis of the backbone dynamics of interleukin -1 beta using two-dimensional inverse detected NMR structure and dynamics of eRF1 middle domain heteronuclear 15 N)1H NMR spectroscopy Biochemistry 29, 7387–74 01 69 Polshakov VI, Birdsall B, Frenkiel TA, Gargaro AR & Feeney J (19 9 9) Structure and dynamics in solution of the. .. angles / and w were derived from the values of 13 Ca, 13 Cb, 13 C¢, 1Ha 1HN and 15 N NMR structure and dynamics of eRF1 middle domain chemical shifts and the software talos [57] Stereospecific assignments for Hbs and pro-R ⁄ pro-S methyl groups of Val and Leu residues, together with the values of torsion angles v1, were obtained using the program anglesearch [58] To generate an initial structure, a set of unambiguously... Tuite MF (19 9 9) The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae Mol Microbiol 32, 485–496 Frolova LY, Merkulova TI & Kisselev LL (200 0) Translation termination in eukaryotes: polypeptide NMR structure and dynamics of eRF1 middle domain 20 21 22 23 24 25 26 27 28 29 30 31 32 release factor eRF1 is composed of functionally and structurally... (200 6) In uence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3 Mol Biol (Mosk) 40, 310 – 316 Hamelberg D & McCammon JA (200 5) Fast peptidyl cis-trans isomerization within the flexible Gly-rich flaps of HIV -1 protease J Am Chem Soc 12 7, 13 778 13 779 Gao F, Mer G, Tonelli M, Hansen SB, Burghardt TP, Taylor P & Sine... release factors eRF1 and eRF3 Cell 12 5, 11 25 11 36 2 Kisselev L, Ehrenberg M & Frolova L (200 3) Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J 22, 17 5 18 2 3 Song H, Mugnier P, Das AK, Webb HM, Evans DR, Tuite MF, Hemmings BA & Barford D (200 0) The crystal structure of human eukaryotic release factor eRF1 ) mechanism of stop codon recognition and peptidyltRNA hydrolysis. .. hydrolysis Cell 10 0, 311 –3 21 4 Kononenko AV, Dembo KA, Kisselev LL & Volkov VV (200 4) Molecular morphology of eukaryotic class I translation termination factor eRF1 in solution Mol Biol (Mosk) 38, 303– 311 5 Bertram G, Bell HA, Ritchie DW, Fullerton G & Stansfield I (200 0) Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition RNA 6, 12 36 12 47 6 Frolova... translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1 Proc Natl Acad Sci USA 99, 8494–8499 Seit-Nebi A, Frolova L & Kisselev L (200 2) Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1 EMBO Rep 3, 8 81 886 Inagaki Y, Blouin C, Doolittle WF & Roger AJ (200 2) Convergence and. .. Montelione GT (19 9 7) Automated analysis of protein NMR assignments using methods from artificial intelligence J Mol Biol 269, 592– 610 51 Ivanova EV, Kolosov PM, Birdsall B, Kisselev LL & Polshakov VI (200 6) NMR assignments of the middle domain of human polypeptide release factor eRF1 J Biomol NMR 36, suppl 1, 8 52 Bax A & Grzesiek S (19 9 3) Methodological advances in protein NMR Acc Chem Res 26, 13 1 13 8 53 Ruckert . ± 11 )9 0 ± 21 )6 4 )4 3 )6 0Lys179 )7 7 ± 13 12 8 ± 12 )6 3 ± 30 )7 0 13 0 )6 0His180 )1 2 8 ± 17 48 ± 68 )1 2 8 ± 93 )1 2 0 45 18 0Gly1 81 80 ± 51 )4 ± 13 90 0Arg182. 0Arg182 )5 3 ± 58 )2 2 ± 46 )6 2 ± 10 5 )6 3 )4 0 )6 0Gly183 )6 6 ± 10 4 )1 3 5 ± 73 )8 7 )1 7 0Gly184 )5 3 ± 44 )2 3 ± 16 )6 3 )3 5Gln185 )9 0 ± 23 13 5 ± 7 )1 1 0 ± 17 )7 5 13 5
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