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Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site Published online 11 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 17 5807–5816 doi:10.1093/nar/gkq344 Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site Eric Ennifar, Philippe Walter and Philippe Dumas* Architecture et Re´ activite´ de l’ARN, Universite´ de Strasbourg, Institut de Biologie Mole´ culaire et Cellulaire du CNRS, 15 rue Rene´ Descartes F-67084 Strasbourg, France Received March 9, 2010; Revised April 15, 2010; Accepted April 19, 2010 ABSTRACT strongly dependent on the three flanking nucleotides (mainly purines) surrounding the self-complementary The crystal structure of subtype-B HIV-1 genomic sequence (4,5). It was shown in vivo that alteration of RNA Dimerization Initiation Site duplex revealed the DIS sequence strongly affects RNA dimerization, chain cleavage at a specific position resulting in packaging and dramatically reduces viral infectivity 0 0 3 -phosphate and 5 -hydroxyl termini. A crystallo- (6–9). In vitro assays have shown that the kissing-loop 2+ 2+ 2+ graphic analysis showed that Ba ,Mn ,Co and complex can be converted into a more stable extended 2+ duplex upon incubation at 55 C, or by the nucleocapsid Zn bind specifically on a guanine base close to the protein at 37 C (10–14) (Figure 1). It has also been shown cleaved position. The crystal structures also point to that kissing-loops formed by the 23-mer DIS RNA used in a necessary conformational change to induce an this study (Figure 1) can be spontaneously converted into ‘in-line’ geometry at the cleavage site. In solution, duplex at 37 C (13,15–17). Such a conversion observed divalent cations increased the rate of cleavage with in vitro with short RNA fragments is invariably presented pH/pKa compensation, indicating that a cation- as accounting for the stabilization of genomic RNA bound hydroxide anion is responsible for the dimers observed during maturation of viral particles cleavage. We propose a ‘Trojan horse’ mechanism, (18). Such an explanation is certainly appealing and plaus- possibly of general interest, wherein a doubly ible but, as far as we know, a formal proof of the occur- charged cation hosted near the cleavage site as a rence in vivo of this often mentioned mechanism is still ‘harmless’ species is further transformed in situ into lacking. an ‘aggressive’ species carrying a hydroxide anion. We have previously solved crystal structures of the HIV-1 subtype-A and -F DIS duplex (19,20), and of subtype-A, -B and -F DIS kissing-complex (21,22). INTRODUCTION These structures revealed unexpected and astonishing structural and sequence similarities between the DIS All retroviral genomes consist in two homologous single dimer and the bacterial 16 S ribosomal RNA aminoacyl stranded RNAs non-covalently linked near their 5 ends. decoding site (A site). Owing to this resemblance, we have Dimerization is an essential step for viral replication. By shown that the DIS tightly bind aminoglycoside antibiot- facilitating template switching of the reverse transcriptase, ics (17,20,23,24). This finding opens interesting dimerization increases recombination and, therefore, vari- structure-based drug design perspectives for targeting spe- ability of the viral genome. The Dimerization Initiation cifically the HIV-1 DIS with aminoglycoside-based mol- Site (DIS) has been identified as a strongly conserved (1) ecules (25,26). stem-loop structure located in the 5 non-coding leader Here, we report the 1.6 A resolution crystal structure of region of the genomic RNA (2,3) (Figure 1). However, the subtype-B DIS extended duplex form. The structure some variations of the nine-nucleotide DIS loop shows some differences compared with HIV-1 subtype-A sequence are tolerated, depending on HIV-1 isolates: and -F duplexes (Supplementary Figure S1). The most A GGUGCACA is mainly found in HIV-1 subtypes 272 280 striking feature is a clear cut in the electron density A and G, A AGCGCGCA in subtypes B and D, 272 280 between G and A showing 5 -hydroxyl and A AGCGCGCU in subtype C and A AGUGCAC 271 272 272 280 272 3 -phosphate termini. The cleavage was also observed in A in subtypes F and H. The loop contains a 6-nt solution and shown to require divalent cations with a self-complementary sequence (underlined) which initiates strong dependence on their ability to downshift the pKa dimerization by forming a loop-loop complex, or ‘kissing- of coordinated water molecules. complex’ (Figure 1). The stability of this complex is *To whom correspondence should be addressed. Tel: +33 388 41 70 02; Fax: +33 388 60 22 18; Email: [email protected] The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 5808 Nucleic Acids Research, 2010, Vol. 38, No. 17 Figure 1. Location and mechanism of HIV-1 RNA dimerization. Schematic drawing of the HIV-1 RNA dimerization mechanism, involving the DIS of two homologous strands (in red and green). The insert shows the subtype-B HIV-1 23-nt DIS fragment used in this study. Changes corresponding to the subtype-A and –F HIV-1 DIS sequence are represented in black and white boxes, respectively. Table 1. Data collection summary and phasing statistics for structure MATERIALS AND METHODS solution RNA synthesis, purification and crystallization 3+ 3+ Native Ru (NH ) Au 3 6 The 23-mer chemically synthesized subtype-B DIS RNA was purchased from Dharmacon and purified using an X-ray source ESRF ID14-2 ESRF BM30 ESRF BM30 ion-exchange Nucleopac PA-100 column as described Wavelength (A) 0.93 0.92 0.92 Max. Resolution (A) 1.60 1.98 2.30 (27). RNA at a concentration of 60 mM was annealed Completeness (%) 99.5 (99.0) 98.9 (99.3) 99.7 (99.9) for 3 min in water at 90 C and cooled to room tempera- Redundancy 9.2 11.2 6.8 ture. It was then incubated for 1 h at 37 C in a crystalliza- a Mean I/s 23.8 (10.0) 53.2 (15.4) 37.0 (10.4) tion buffer (20 mM Na cacodylate pH 7.0, 5 mM MgCl , 2 R (%) 6.3 (24.0) 6.9 (13.5) 4.2 (10.4) sym 300 mM KCl) and concentrated to 500–600 mM. Phasing power centric 1.0 0.87 – Crystallization was performed in sitting drop by adding acentric (iso/ano) 1.0/– 1.0/– /2.5 one volume of crystallization solution made with MPD R (centric) 0.89 0.84 – cullis (20%) and spermine (50 mM) to nine volumes of RNA ˚ ˚ ˚ in the crystallization buffer. Drops were equilibrated at Space group: P2 2 2; unit cell a = 44.0 A, b = 47.5 A, c = 57.7 A. Mean 1 1 figure of merit before/after solvent flattening: 0.38 (0.07)/0.86 (0.80). 37 C with a reservoir made with 50% MPD, 50 mM Na Values in parenthesis are those for outermost shell. cacodylate, 100 mM MgCl and 300 mM KCl. Platelets 2 b Phasing power = <F /LOC>, where LOC is the lack of closure. crystals appeared within 4–10 days and were stabilized at c R =| |F ± F |– F |/|F – F | for centric reflections. cullis PH P H PH P 20 C and cryo-protected by soaking in reservoir solution. Table 2. Data collection summary for divalent metal-soaked Crystal soaking, X-ray data collection and processing structures Heavy atom derivatives for the MIR method were 2+ 2+ 2+ 2+ obtained by soaking crystals for several days in a reservoir Mn Zn Co Ba solution containing 25 mM ruthenium(III) hexamine X-ray source ESRF ESRF Rot. anode Rot. anode (without magnesium) or 10 mM AuCl (in presence of BM30 BM30 100 mM MgCl ). Soaking of crystals was also performed 2 ˚ Wavelength (A) 1.41 1.28 1.54 1.54 for the determination of the binding sites of cations Max. Resolution (A) 2.60 1.90 2.35 2.56 Completeness (%) 98.4 (92.7) 87.0 (41.9) 94.2 (94.6) 99.2 (94.5) inducing cleavage. Daylong soaking of the crystals in 2+ 2+ Redundancy 3.3 4.8 2.4 5.6 100 mM Mn or Ba was possible without affecting Mean I/ 21.1 (12.0) 19.7 (10.1) 14.1 (2.2) 27.5 (12.4) their diffraction power; for zinc and cobalt, it was neces- R (%) 6.0 (8.3) 6.8 (8.5) 6.1 (38.2) 6.5 (13.2) sym sary to reduce salt concentration to 20 mM and the soaking time to 1 h. After stabilization, crystals were Values in parentheses are those for outermost shell. flash-cooled in liquid ethane (simple flash cooling under the nitrogen gas stream usually resulted in a serious loss of diffraction). All data were collected at 100 K (Tables 1 were localized by Fourier-difference maps. Phasing was and 2), and processed using the HKL package (28). initiated at 2.0 A resolution with SHARP 3.0 (31) using the gold derivative, and not the native, as the reference Structure solution and refinement of structures dataset. The figure of merit was greatly improved by Two strong heavy atom sites were localized using anom- solvent flattening with SOLOMON (32) using a solvent alous differences with LOCHVAT (29,30) for the gold content of 40%. The resulting experimental map was of derivative. Subsequently, three ruthenium-binding sites excellent quality, revealing one dimer per asymmetric unit Nucleic Acids Research, 2010, Vol. 38, No. 17 5809 packed along the c axis so as to form pseudo-infinite the results show a very similar pattern at the two pH helices. The model was built with O 6.2 (33) and structures values (Figure 5b) may be viewed as an indication that of pre-cleaved and post-cleaved were refined with CNS the procedure was effective. All calculations, comprising (34) (Table 3). Potassium, zinc, cobalt and manganese image gel processing, were made with Mathematica from ions were identified using anomalous Fourier-difference Wolfram Research. maps as described (19). Analysis of RNA cleavage in solution RESULTS The 23-mer DIS RNA at a concentration of 90 mMin Description of the structure water was heated at 90 C for 5 min. and slowly cooled to room temperature for several hours to allow duplex As observed in previously described crystal structures of formation. The sample was then diluted to 12 mMina DIS extended duplex, the subtype-B DIS extended duplex buffer containing 150 mM potassium acetate, 20 mM structure looks quite similar to its kissing-complex coun- sodium cacodylate pH 6.85 or pH 6.31, and 5 or 20 mM terpart (21,22) in spite of the different RNA topology of divalent cations (MgCl , MnCl , ZnCl , CoCl , BaCl , (Supplementary Figure S2). The structure is mainly 2 2 2 2 2 Pb(II) acetate) or 3 mM EDTA, and incubated at 37 C. characterized by A and A of both strands related 272 273 Lead acetate was used for its higher solubility in compari- by non-crystallographic symmetry (named strands a and son of lead chloride. Slightly acidic conditions prevented b in the following) being extrahelical and forming a the formation of insoluble hydroxides. For visualization four-base stack, the A of each strand remaining of the 5 -cleavage product and quantification, unpaired inside the helix (Figure 2). A comparison of 0 32 5 [g P]ATP-labelled RNA was mixed with unlabelled these flanking purines among various DIS crystal struc- RNA. The RNA was analysed on denaturing 8 M urea, tures are described in Supplementary Data 20% polyacrylamide gel electrophoresis (DPAGE) in TBE (Supplementary Figure S3). In spite of a local phosphate buffer (45 mM tris–borate pH 8.3, 2 mM EDTA) and the backbone distortion induced by the inter-strand adenine cleavage position was checked using RNase T1 and stacking, the duplex remains perfectly linear (Figure 2b). alkaline ladders. Gel quantification was made with a In the present structure, A and A are involved in 273a 273b Fuji phosphoimager. Raw data from the phosphoimager ‘A-minor’ (35,36) contacts with the minor groove of a software were obtained as binary image files. To trans- symmetry-related duplex molecule, leading to an inter- form the raw value at each pixel of the image into a action very similar to one described in the 30 S ribosomal value proportional to the radioactivity count, the follow- subunit [see Figure 7b in (37)]. It may be hypothesized that 4RawValue ing transformation RawValue ! 10 (unim- such an interaction occurs within the complete portant pre-exponential factors are omitted) was initially applied, according to information from the manufacturer. We found it necessary to refine the latter transformation as 3:86RawValue RawValue ! 10 to fit correctly the response obtained from a known dilution scale. When the 2+ cleavage was fast enough, as for Pb , the cleavage rate constants k could have been derived following the relation kt f ¼ 1  e where f is the fraction of cleaved RNA obs obs (Supplementary Figure S13b). However, in situations of 2+ 2+ 2+ slow and incomplete cleavage, as for Co ,Mn or Mg , the inescapable variations in the amount of radioactive materials loaded in each lane of the gel made this proced- ure too inaccurate. An internal standardization was thus used within each lane by considering also the small-size products (Supplementary Figures S10 and S13a). The pro- cedure is explained in Supplementary Data. The fact that Table 3. Refinement statistics 2+ 2+ Native Mn soaked Co soaked Max. resolution (A) 1.6 2.6 2.4 Completeness (work + test sets) 95.4% 97.7% 91.2% R factor 25.0% 23.0% 28.4% free R factor 24.2% 20.0% 23.9% Estimated coordinate errors (A) 0.22 0.34 0.36 Average B factor (A ) 32.0 20.4 42.0 Figure 2. Structure of the HIV-1 subtype-B DIS extended duplex. The Number of nucleic acid atoms 982 982 982 two strands are represented in green and red. The black arrowhead Number of water molecules 183 149 46 depicts the asymmetric cleavage site observed in crystals. (a) Number of metal atoms 5 8 5 Sequence and secondary structure of the duplex. (b) Stereo view of Protein Data Bank ID 2OIY 2OJ0 3FAR the structure solved in this study. 5810 Nucleic Acids Research, 2010, Vol. 38, No. 17 encapsidation region in the viral RNA after dimerization conformations of G ), no significant cleavage could be 271a and maturation of virions (38,39). detected at G on the other strand related by 271b non-crystallographic symmetry. In agreement with this The subtype-B DIS duplex is cleaved in crystals and in observation the ribose of G clearly appeared in the 271b solution C2 -endo conformation as predominantly observed in absence of cleavage at that stem terminal base-pair The most striking feature of the subtype-B duplex struc- (21,22). Altogether, these observations are strongly in ture is the presence of a cleavage between G and A of 271 272 favour of the existence of cation-dependent cleavage in one of the two strands (strand a in green on Figure 2b). the crystals. This obviously does not exclude that a part The cleavage was first revealed during refinement of the of the RNA molecules integrated in a crystal were previ- structure by inspection of electron density maps from ously cleaved in solution. several crystals. It appeared on (Fo–Fc) difference Fourier maps as a strong positive peak close to ribose Crystallographic study of cation binding near 271a, which corresponds to the position of the phosphate the cleavage site after cleavage, and as a strong negative peak on its expected position before cleavage (Figure 3a). In agree- To understand the role of metal ions in the primary ment with this interpretation, a composite simulated an- cleavage mechanism, we soaked crystals for several days nealing omit map showed a very poor density for the with magnesium-free solutions containing either manga- ribose of G in a C2 -endo conformation, and for the nese, zinc, barium or cobalt divalent cations. Anomalous 271a G -A phosphodiester bond. The cleavage was con- difference maps for manganese, zinc and cobalt (for 271a 272a firmed by a polyacrylamide gel analysis of RNA from barium, see below) revealed a strong peak in the vicinity dissolved crystals and from drops where they had grown of G for manganese [11.5 estimated standard devi- 271a (Figure 3b). Furthermore, inspection of various electron ations (e.s.d.)], zinc (19.5 e.s.d.) and cobalt (8.9 e.s.d.) density maps clearly showed an alternative C3 -endo con- (Figure 4a and Supplementary Figures S5 and S6). In formation of the G ribose and a 3 -phosphate resulting addition, simulated annealing composite omit maps 271a from cleavage. This suggested that cleavage proceeds showed a conformational change of the sugar–phosphate through the classical mechanism of nucleolytic ribozymes backbone between residues 273a and 274a, resulting in an by a two-step reaction involving first a trans-esterification inward rotation of phosphate 274a and an outward 2.3 A 0 0 producing strand scission and a 2 -3 -cyclic phosphate translation movement of G (Figure 4b). This local con- 274a intermediate and, second, followed by its hydrolysis re- formational change creates a negatively charged metal ion sulting in the 3 -phosphate product [Supplementary pocket, where the partially dehydrated divalent ion is Figure S4; for review, see (40)]. Such a mechanism was directly bound to N7 of G , and to anionic oxygen 274a first observed for the tRNA(Phe) (41,42), and for atoms of phosphates 273a and 274a (Figure 4a). This con- lead-dependent ribozymes (43), but not with hammerhead, trasts with the catalytic divalent cation bound by hairpin and hepatitis d ribozymes that are unable to outer-sphere coordination to the active site of the HDV 0 0 catalyse hydrolysis of the 2 -3 -cyclic phosphate ribozyme (46). Notably, the residues surrounding G , 274a produced by the first trans-esterification [for review, see including the bulged adenines, are not affected by this (44,45)]. motion, and the base pair G -C is not disrupted 274a 279b Surprisingly, whereas one strand in the crystals was (Figure 4a). Although the cation binding scheme is 2+ 2+ cleaved to within 30–40% (which was estimated by similar for Mn and Zn , the distance from each refining the occupancy of the pre- and post-cleaved cation to the 2 -OH group of the cleaved G residue 271a Figure 3. Cleavage observed in the HIV-1 subtype-B extended duplex. (a) Stereo view of the cleaved region. The model used for electron density map calculations corresponds to the uncleaved form. (3Fo–2Fc) electron density map contoured at 1.4 e.s.d. is shown in blue; (Fo–Fc) difference map is shown contoured at 5.0 e.s.d. (red), and –3.5 e.s.d. (orange). The latter shows an excess of density on phosphate 272 as built in the uncleaved model (negative peak), and a lack of density (positive peak) corresponding to its position after the cleavage. (b) Denaturing PAGE analysis of RNA extracted from crystallization drop, from a dissolved crystal, and before crystallization (0). Only the largest 5 -fragment resulting from cleavage is visible upon ethidium bromide staining. Nucleic Acids Research, 2010, Vol. 38, No. 17 5811 Figure 4. Cation-induced conformational change at the A –G step. (a) Stereo view of the composite simulated annealing omit map contoured at 273 274 1.4 e.s.d. showing a clear density for residues A and G after the conformation change (indicated with black arrows), without disrupting the 273a 274a G –C base-pair. The anomalous-difference map corresponding to the manganese ion is represented in orange and contoured at 8.0 e.s.d. The 274a 281b ˚ ˚ ˚ cation lies at 2.4 A from the N7(G ) and at 2.3 A and 2.0 A from each phosphate. (b) Superimposition of the native (in gray) and the 274a 2+ 2+ 2+ manganese-soaked structures (in blue). The binding of Mn ,Co or Zn induces a 2.3 A movement of base and ribose 274, as well as a rotation of phosphate 273 toward the interior of the helix (pink circled region). 2+ 2+ ˚ ˚ varies from 6.5 A for Mn to 5.6 A for Zn . For the Two strong peaks in an anomalous difference map (13.2 2+ latter cation, the anomalous difference map displays a and 11.5 e.s.d.) were also seen with Ba on G and 274a G (Supplementary Figure S7). At variance with other major peak and also, well above the background, an 274b 2+ cations, Ba is shifted toward O6 of G and G , elongated extension which may be the mark of a more 274a 274b which prevents it from a direct coordination with phos- complex binding mode (Supplementary Figure S5). phates 273 and 274. Also, no cleavage was visible with Interestingly, no significant peak was detected in anomal- 2+ 2+ 2+ Ba at G and some cleavage was visible at G , 271b 271a ous difference map for Zn and Mn on G on the 274b but very likely this cleavage was due to the presence of other strand and, concomitantly, the electron density map 2+ 2+ Mg prior to soaking with Ba . Such a peculiarity did not reveal any cleavage at G . 271b 2+ will be rationalized fully in the following. Finally, this For Co , on the contrary, a strong peak (7.4 e.s.d.) was cation binding study also showed, as previously reported also found in an anomalous difference map on G and, 274b (27), that metal ion binding to RNA can be very sensitive concomitantly, a cleavage at G was observed 271b 2+ 2+ to small structural variations since only Ba and Co (Supplementary Figure S6). This confirms that cleavage were able to bind at the two a priori equivalent sites can take place in the crystals and that divalent cations 2+ G and G . Such a non-symmetric binding for 274a 274b are involved in the cleavage. The binding mode of Co 2+ 2+ 2+ 2+ Mn and Zn is likely limited to the crystalline state also differs from Zn and Mn because, first, no inward and, therefore, both strands should be susceptible to movement of phosphate 274 is observed, which results in a cleavage in solution. direct coordination of the cation only to N7 of G and, 2+ 0 second, because the distances between Co and 2 -OH Solution study of the cleavage groups of G and G (4.8 and 3.8 A) are shorter 274a 274b 2+ than for their Zn counterparts (Supplementary Figure Although the involvement of the G 2 -OH group in the S6). It is difficult to decide whether these differences are cleavage reaction was extremely probable, we confirmed it related to the different nature of each cation, to enhanced by using a mutated DIS sequence where this residue was cleavage in the crystal, or to both. replaced by a deoxyguanosine; as expected, cleaving 5812 Nucleic Acids Research, 2010, Vol. 38, No. 17 activity was abolished (Supplementary Figure S8). We correlates very well with their shifting of the pKa of 2+ 2+ 2+ 2+ then compared the ability of Mg ,Mn ,Zn ,Co , coordinated water molecules since we obtained 2+ 2+ 2+ Pb ,Ba and Ca to induce strand cleavage in  log k=pKa ¼1:06  0:1 at pH 6.30 and solution. Experiments were performed at 37 C and result-  log k=pKa ¼0:93  0:14 at pH 6.85, where k is the ing fragments were separated by denaturing PAGE. cleavage rate constant (Figure 5b). Therefore, Ethidium bromide staining revealed the 16-mer fragment  log k=pKa ¼1 within experimental errors. We also corresponding to the 3 cleavage product. This also observed that lowering the pH by 0.55 unit (from 6.85 to revealed a distinct band, which was unexpected since 6.3) resulted in less cleavage for all cations with a mean only one cleavage site could be seen in the crystal structure value  log k=pH ¼ 0:98  0:16 (Figure 5b). Thus, (Figure 5a). We note that this additional band was absent within experimental errors, there exists a pKa/pH com- 2+ with Co . Essentially, this was the mark of an additional pensation on the kinetics of cleavage, which implies that cleavage site at A280, opposite to the cation binding site. a cation coordinated to a hydroxide ion is responsible for More details about this are in Supplementary Data. In the the activation of the 2 -OH group of G leading to 271a following, the cleavage at G271 and at A280 will be cleavage. Indeed, considering the acid-base equilibrium 2+ 2+ referred to, respectively, as the primary and the secondary between the two species M (H O) and M (H O) 2 n 2 n–1 pHpK pHpK a a cleavage cleavage site. OH yields m ¼ m 10 =ð1+10 Þ, where m is the Under conditions close to those used for crystallization concentration of the cation bearing a hydroxide anion and 2+ (20 mM Mg ), the primary cleavage is rather slow m is the total cation concentration. This accounts well for compared to small RNA ribozymes. Only 7% of the the observed pKa/pH compensation. If the active cationic species were not to be bound to the RNA was cleaved after 14 h of incubation (Figure 5), 5 1 which yields a rate constant k 2+= 8.6  10 min .In RNA at some stage prior cleavage, one would expect that, Mg presence of either 3 mM EDTA and 100 mM Na ,or of at a given pH, the rate of cleavage should be proportional saturating concentration of cobalt(III) hexammine, no to the total cation concentration m . However, with man- cleavage was detectable even on daylong time-scale, con- ganese, for which accurate data could be obtained at both firming that divalent metal ions are required for the pH, a 4-fold increase of total concentration from 5 to reaction (Supplementary Figure S9 and not shown). We 20 mM yielded k(20 mM)/k(5 mM) = 2.2 ± 0.15 at have thus compared the efficiency of cleavage with differ- pH 6.85 and k(20 mM)/k(5 mM) = 2.4 ± 0.5 at pH 6.30, 2+ 2+ 2+ 2+ 2+ 2+ ent cations: Ca ,Ba ,Mn ,Co ,Zn and Pb which are values significantly different from 4. Such a (Figure 5 and Supplementary Figures S9, S10 and S11). value close to 2.3 agrees well with the need of cation Cleavage rates observed for the DIS duplex are compar- binding to the RNA before cleavage if the binding of 2+ able to those observed for the secondary cleavage sites in Mn at 20 mM is in excess to its binding at 5 mM pre- the minimal hammerhead ribozyme (47,48). In our case, cisely by a factor 2.3. For this to take place the dissoci- 2+ 2+ the most efficient cation is Pb , which led to 42% of ation constant of Mn has to be K = 9 mM. 2+ Interestingly, a much more detailed analysis of the same cleavage after 1 h at 5 mM concentration (k = Pb 2 1 2+ 2+ kind was performed for the binding of magnesium to the 10 min ), and the less efficient are Ca and Ba , which did not promote any detectable cleavage after 14 h HDV ribozyme and led to quite comparable values (Figure 5a and data not shown). (K = 3.1 mM at pH 6.5) (49). Furthermore, our value Analysis of the cleavage rates showed that the ranking K = 9 mM fits also well with apparent dissociation con- of the tested cations for their ability to cleave the duplex stants of various divalent cations (from 5 to 12 mM) that Figure 5. Analysis of the cleavage of 23-mer subtype-B DIS duplex in solution. (a) Metal ion dependency of the cleavage at pH 6.85 and 37 C. The 2+ cleavage with Zn was also performed at pH 6.3. (b) Variation at two pH values of the cleavage rate versus the pKa of each cation at 5 mM. The 2+ kinetic constant could not be determined at the lower pH for Mg . The error bars are those from the numeric procedure exposed in Supplementary Data and are not fully representative of the overall errors which are certainly more important (see text). Nucleic Acids Research, 2010, Vol. 38, No. 17 5813 were reported for a modified Schitosoma hammerhead ribozyme (50). Finally, we also performed competition ex- 2+ 2+ periments with Ba and Ca that are inefficient at 2+ 2+ cleaving the RNA. As expected, Ba as well as Ca in- 2+ hibited the Co -dependent cleavage (Supplementary 2+ Figure S12). This is consistent with Co being dislodged from the specific binding sites by competitive inhibitors (albeit we have a formal crystallographic proof of 2+ specific binding only for Ba ; see Supplementary 2+ Figure S7). Half inhibition by Ba was seen to take place roughly in the range 6–30 mM (Supplementary Figure S12). Calculation of the inhibition factor shows 2+ that this is explained if the K of Ba is comprised between 5 and 25 mM, which seems also reasonable in view of the values just mentioned. Although competitive inhibition agrees with our results, one cannot exclude that, 2+ 2+ at the highest Ba or Ca concentrations, electrostatic screening too (51), and not only direct competition for the crucial binding sites, was responsible for cleavage inhibition. DISCUSSION Proposition of a detailed cleavage mechanism By using the different views of the cleavage site provided by our crystal structures, as well as the results from solution studies, we can propose the following cleavage mechanism for the DIS duplex (Supplementary Figure S4). Clearly, the rather slow cleaving rate observed in this structure compared to ‘standard ribozymes’ is an ad- vantage for crystallographic studies by allowing the trapping of intermediate states. First, a snapshot of the structure before cleavage is provided by the uncleaved strand, which shows ribose 271 in a C2 -endo conform- ation (Figure 6a). Second, there would be a necessary intermediate consisting in the binding of a divalent cation with a suitable pKa at the A –G step; depend- 273 274 ing on the cation, there is a local rearrangement leading to the involvement of A ,G phosphates in the binding 273 274 (Figure 6b). The metal ion would then be able to provide a Figure 6. Proposition for a cleavage mechanism. (a) Snapshot of the hydroxide anion accepting a proton from the 2 -OH group initial state provided by the uncleaved strand. Residue G is in a C2 - of G . Third, the cleavage reaction would require a endo conformation and no metal ion is present in the pocket, which is closed by an unusual CH8(G )-phosphate(A ) hydrogen bond. The 274 273 crankshaft motion of the A phosphate towards the (3Fo–2Fc) electron density map contoured at 1.4 e.s.d. is shown in blue. interior of the helix to obtain a configuration of the A The gray arrowhead shows the cleavable phosphodiester bond. (b)A 0 0 2+ scissile bond in line with the O2 of G in a C2 -endo divalent metal ion (here Mn ) binds at the A –G step, inducing a 273 274 conformation. A model of such a conformation is shown rearrangement of the sugar-phosphate backbone. The anomalous-difference map contoured at 8.0 e.s.d. is shown in orange. in Figure 6c. This unstable phosphate conformation, very (c) The metal ion reduces the pKa of a bound water molecule and likely the chemical limiting step, was not observed in our generates hydroxide anions that activate the 2 -OH group of the G crystal structure, but would be very similar to the one ribose. A necessary movement of the phosphate 272 toward the interior observed at the cleavage site of the fully active extended of the helix (curved arrow #1) then orients the cleavable bond in-line hammerhead ribozyme (52). Such an in-line conformation with the activated 2 -hydroxyl group thus allowing (curved arrow #2) a 0 0 nucleophilic attack leading to a 2 -3 -cyclic phosphate intermediate. was also observed at a secondary cleavage site of the This product is not stable in crystals and is hydrolyzed (d) into a minimal hammerhead ribozyme where it was stabilized 3 -monophosphate (in red) product observed in this simulated anneal- by a direct interaction with a zinc cation (48) ing composite omit map (contoured at 1.4 e.s.d.). (Supplementary Figure S13). Such a 2 -OH-mediated 3 -phosphate product as previously observed with the nucleophilic attack can only result in strand scission 0 0 0 leadzyme (43). In the absence of a cyclic phosphate the with a 2 -3 -cyclic phosphate and 5 -OH termini. Since 0 0 the 2 -3 -cyclic phosphate could not be detected in the G ribose is no more constrained and is free to switch 0 0 electron density map, this means that it should be from its initial C2 -endo to a C3 -endo conformation rec- hydrolyzed quickly enough to yield the observed ognizable in electron density maps (Figure 6d). 5814 Nucleic Acids Research, 2010, Vol. 38, No. 17 Clearly, one conflicting observation should be ad- A Trojan horse mechanism? 2+ dressed. Indeed, there is a difference between Mg , Since cation binding is a prerequisite for cleavage and which was not visible in the electron density map and since the active species corresponds to a partially yet is able to cleave (since the original observation of neutralized cation carrying a hydroxide anion, a doubly 2+ cleavage was in a Mg -containing crystal), and other charged cation should be a strong inhibitor of the cleavage cleaving cations that were visible in electron density for two reasons. First, this is because its concentration is 2+ maps. This is not unprecedented since the catalytic Pb higher than that of the active species; for zinc for example, in the leadzyme ribozyme for example (53) could not be 2+ it can be estimated from (56) that [Zn ] is in 20-fold 2+ localized in crystal structures. In the present case, we have excess versus [Zn OH ]. Secondly, the affinity for the 2+ no clear-cut explanation for this difference between Mg anionic binding site of the doubly charged species is cer- and other cations. Nevertheless, it may be mentioned that tainly also much higher than that of the singly charged 2+ Mg (10 electrons) is inherently more difficult to observe species. It is thus tempting to suggest a scenario in two 2+ than the other cations we used (from 23 to 80 electrons). steps. First, such an inactive doubly charged M species Furthermore, magnesium has an exceedingly small anom- would indeed bind, and well, but without being protected alous scattering component, which makes useless an against the H O/OH exchange. Second, once the cation 2+ anomalous difference map to reveal its presence in a has been partially neutralized as M (OH ), and thus 2+ specific binding site, if any. Finally, Mg might also transformed in situ into the active singly charged species, 2+ 2+ 2+ bind less strongly than Mn ,Co and Zn , but suffi- it would be also more easily released in the immediate 2+ ciently to provoke cleavage, and/or Mg is released after vicinity of the cleavable site. Expressed in a metaphoric 2+ 2+ 2+ cleavage more easily than Mn ,Co and Zn . Either way, the cleavage would result from some ‘Trojan horse’ possibility is consistent with the stronger affinity for N7 mechanism wherein an apparently ‘inoffensive’ cation 2+ atom of the latter cations that are softer than Mg [in this species is hosted next to the cleavage site but, soon or respect, see (54)]. later, is transformed into a more easily released ‘aggres- It is interesting to compare the cleavage pattern sive’ species. The interesting consequence is that the more observed in the DIS RNA after cation binding at a tightly bound an inhibitor is, the more chance it has to be specific site with an artificial system using lanthanide transformed in situ into the aggressive species. Such an complexes covalently linked to an oligoDNA hybridized interpretation should be of general extent in situations to an oligoRNA (55). When a perfect helical duplex where a bound cation is active as the general base (as, was formed, due to full complementarity of the RNA we think, this holds true in the present case). and DNA sequences, a cleavage was observed, Comparison with other cation-dependent ribozyme but only at the phosphodiester bond opposite to the nucleotide bearing the lanthanide complex. Interestingly, This DIS fragment should be viewed as a metal-dependent the position opposite to the cation binding site in the DIS ribozyme, but certainly not as a very efficient one. There is A280 (Figure 2), where a secondary cleavage was are two reasons for that. First, the necessary conform- also observed (Supplementary Figures S10 and S11). ational change yielding the in-line geometry (Figure 6c) When the artificial system was designed with an imperfect requires a high, and may be even a very high, activation sequence complementarity allowing an RNA bulge to energy. Second, we did not detect any candidate to act as form in the vicinity of the lanthanide complex, up the general acid providing a proton to the leaving group. to four additional consecutive cuts were observed in Very likely, this role is played by a water molecule from the stretch of unpaired residues forming the bulge. the bulk solvent. On the contrary, in very efficient ribo- Overall, the latter observation is strikingly similar zymes, the structure is tuned for a favourable orientation to what we observed with the DIS, the primary cleavage of the cleavable bond and, furthermore, both the general site being at G271 next to the extrahelical purines A272, base and the general acid are identified as pre-structured A273 corresponding to the bulged-out residues in entities. This is the case, for example, for the hepatitis the artificial system. However, in our case, a more delta virus (HDV) ribozyme where a bound magnesium specific pattern was seen with cobalt which produced no cation and a cytosine base (C75) were clearly identified. significant cleavage at A280 (opposite to the cation However, somewhat strangely, no definite consensus binding site) and almost no additional cleavage surround- emerged about which acts as the general acid, and which ing the major cleavage site at G271 (Supplementary acts as the general base (46,57–59). A ‘multichannel Figure S11). This is certainly related, albeit not in an reaction mechanism’, instead of a presupposed unique obvious way, to the previously mentioned different mechanism, could reconcile the opposite views (49). binding mode of cobalt versus zinc and manganese. Recently, a new metal ion binding site was described at Therefore, what this comparison shows is that the gross a position that is consistent with a magnesium-bound hy- features of the cleavage pattern in the DIS do not seem droxide serving as a general base (60). This would there- to derive from the genuine specificity of binding of fore be comparable to our observations. divalent cations, but rather from the cation binding site In the case of the DIS ribozyme, although the role of the being close to the covalent bond susceptible to cleavage cation as the general base seems established, it is unclear (see the ‘Conclusion’ section). Some peculiarities, whether the hydroxide anion has to be shuttled by the however, do result from the specificity of binding, as cation to the accepting hydroxyl group, or whether it seen with cobalt. may simply be released by the cation close to it. In the Nucleic Acids Research, 2010, Vol. 38, No. 17 5815 6. Paillart,J.-C., Berthoux,L., Ottmann,M., Darlix,J.-L., Marquet,R., first case, the cation would be directly involved into the Ehresmann,C. and Ehresmann,B. (1996) A dual role of the chemical step (possibly interacting also with the A272 dimerization initiation site of HIV-1 in genomic RNA packaging phosphate after its conformational change (Figure 6c), and proviral DNA synthesis. J. Virol., 70, 8348–8354. which would stabilize the transition state), whereas in 7. Berkhout,B. and van Wamel,J.L. (1996) Role of the DIS hairpin the second case it would act at a distance. In this in replication of human immunodeficiency virus type 1. J. Virol., 70, 6723–6732. respect, a thorough comparison with protein enzymes 8. Clever,J.L. and Parslow,T.G. (1997) Mutant HIV-1 genomes with has led to the proposal that a cation can indeed exert a defects in RNA dimerization or encapsidation. J. Virol., 71, catalytic effect without contacting directly its ‘target’ (61). 3407–3414. This is certainly not against the previous conclusion drawn 9. Laughrea,M., Jette´ ,L., Mak,J., Kleinman,L., Liang,C. and Wainberg,M.A. (1997) Mutations in the kissing-loop hairpin of from the comparison with the artificial system using lan- human immunodeficiency virus type 1 reduce viral infectivity as thanide complexes (55). well as genomic RNA packaging and dimerization. J. Virol., 71, 3397–3406. 10. Laughrea,M. and Jette´ ,L. (1996) Kissing-loop model of HIV-1 ACCESSION NUMBERS genome dimerization: HIV-1 RNA can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin Atomic coordinates and structure factors of the native, the 248-271 are dispensable for dimer formation. Biochemistry, 35, manganese- and the cobalt-soaked structures have been 1589–1598. 11. Muriaux,D., Fosse´ ,P. and Paoletti,J. (1996) A kissing complex deposited with the Protein Data Bank (ID 2OIY, 2OJ0 together with a stable dimer is involved in the HIV-1 RNA Lai and 3FAR, respectively). dimerization process in vitro. Biochemistry, 35, 5075–5082. 12. Muriaux,D., Rocquigny,H.D., Roques,B.P. and Paoletti,J. (1996) NCP7 activates HIV-1 Lai RNA dimerization by converting a SUPPLEMENTARY DATA transient loop-loop complex into a stable dimer. J. Biol. Chem., 271, 33686–33692. Supplementary Data are available at NAR Online. 13. Takahashi,K.I., Baba,S., Chattopadhyay,P., Koyanagi,Y., Yamamoto,N., Takaku,H. and Kawai,G. (2000) Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome. RNA, 6, 96–102. ACKNOWLEDGEMENTS 14. Takahashi,K.I., Baba,S., Koyanagi,Y., Yamamoto,N., Takaku,H. The authors thank Philippe Carpentier for his abundant and Kawai,G. 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Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

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Abstract

Published online 11 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 17 5807–5816 doi:10.1093/nar/gkq344 Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site Eric Ennifar, Philippe Walter and Philippe Dumas* Architecture et Re´ activite´ de l’ARN, Universite´ de Strasbourg, Institut de Biologie Mole´ culaire et Cellulaire du CNRS, 15 rue Rene´ Descartes F-67084 Strasbourg, France Received March 9, 2010; Revised April 15, 2010; Accepted April 19, 2010 ABSTRACT strongly dependent on the three flanking nucleotides (mainly purines) surrounding the self-complementary The crystal structure of subtype-B HIV-1 genomic sequence (4,5). It was shown in vivo that alteration of RNA Dimerization Initiation Site duplex revealed the DIS sequence strongly affects RNA dimerization, chain cleavage at a specific position resulting in packaging and dramatically reduces viral infectivity 0 0 3 -phosphate and 5 -hydroxyl termini. A crystallo- (6–9). In vitro assays have shown that the kissing-loop 2+ 2+ 2+ graphic analysis showed that Ba ,Mn ,Co and complex can be converted into a more stable extended 2+ duplex upon incubation at 55 C, or by the nucleocapsid Zn bind specifically on a guanine base close to the protein at 37 C (10–14) (Figure 1). It has also been shown cleaved position. The crystal structures also point to that kissing-loops formed by the 23-mer DIS RNA used in a necessary conformational change to induce an this study (Figure 1) can be spontaneously converted into ‘in-line’ geometry at the cleavage site. In solution, duplex at 37 C (13,15–17). Such a conversion observed divalent cations increased the rate of cleavage with in vitro with short RNA fragments is invariably presented pH/pKa compensation, indicating that a cation- as accounting for the stabilization of genomic RNA bound hydroxide anion is responsible for the dimers observed during maturation of viral particles cleavage. We propose a ‘Trojan horse’ mechanism, (18). Such an explanation is certainly appealing and plaus- possibly of general interest, wherein a doubly ible but, as far as we know, a formal proof of the occur- charged cation hosted near the cleavage site as a rence in vivo of this often mentioned mechanism is still ‘harmless’ species is further transformed in situ into lacking. an ‘aggressive’ species carrying a hydroxide anion. We have previously solved crystal structures of the HIV-1 subtype-A and -F DIS duplex (19,20), and of subtype-A, -B and -F DIS kissing-complex (21,22). INTRODUCTION These structures revealed unexpected and astonishing structural and sequence similarities between the DIS All retroviral genomes consist in two homologous single dimer and the bacterial 16 S ribosomal RNA aminoacyl stranded RNAs non-covalently linked near their 5 ends. decoding site (A site). Owing to this resemblance, we have Dimerization is an essential step for viral replication. By shown that the DIS tightly bind aminoglycoside antibiot- facilitating template switching of the reverse transcriptase, ics (17,20,23,24). This finding opens interesting dimerization increases recombination and, therefore, vari- structure-based drug design perspectives for targeting spe- ability of the viral genome. The Dimerization Initiation cifically the HIV-1 DIS with aminoglycoside-based mol- Site (DIS) has been identified as a strongly conserved (1) ecules (25,26). stem-loop structure located in the 5 non-coding leader Here, we report the 1.6 A resolution crystal structure of region of the genomic RNA (2,3) (Figure 1). However, the subtype-B DIS extended duplex form. The structure some variations of the nine-nucleotide DIS loop shows some differences compared with HIV-1 subtype-A sequence are tolerated, depending on HIV-1 isolates: and -F duplexes (Supplementary Figure S1). The most A GGUGCACA is mainly found in HIV-1 subtypes 272 280 striking feature is a clear cut in the electron density A and G, A AGCGCGCA in subtypes B and D, 272 280 between G and A showing 5 -hydroxyl and A AGCGCGCU in subtype C and A AGUGCAC 271 272 272 280 272 3 -phosphate termini. The cleavage was also observed in A in subtypes F and H. The loop contains a 6-nt solution and shown to require divalent cations with a self-complementary sequence (underlined) which initiates strong dependence on their ability to downshift the pKa dimerization by forming a loop-loop complex, or ‘kissing- of coordinated water molecules. complex’ (Figure 1). The stability of this complex is *To whom correspondence should be addressed. Tel: +33 388 41 70 02; Fax: +33 388 60 22 18; Email: [email protected] The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 5808 Nucleic Acids Research, 2010, Vol. 38, No. 17 Figure 1. Location and mechanism of HIV-1 RNA dimerization. Schematic drawing of the HIV-1 RNA dimerization mechanism, involving the DIS of two homologous strands (in red and green). The insert shows the subtype-B HIV-1 23-nt DIS fragment used in this study. Changes corresponding to the subtype-A and –F HIV-1 DIS sequence are represented in black and white boxes, respectively. Table 1. Data collection summary and phasing statistics for structure MATERIALS AND METHODS solution RNA synthesis, purification and crystallization 3+ 3+ Native Ru (NH ) Au 3 6 The 23-mer chemically synthesized subtype-B DIS RNA was purchased from Dharmacon and purified using an X-ray source ESRF ID14-2 ESRF BM30 ESRF BM30 ion-exchange Nucleopac PA-100 column as described Wavelength (A) 0.93 0.92 0.92 Max. Resolution (A) 1.60 1.98 2.30 (27). RNA at a concentration of 60 mM was annealed Completeness (%) 99.5 (99.0) 98.9 (99.3) 99.7 (99.9) for 3 min in water at 90 C and cooled to room tempera- Redundancy 9.2 11.2 6.8 ture. It was then incubated for 1 h at 37 C in a crystalliza- a Mean I/s 23.8 (10.0) 53.2 (15.4) 37.0 (10.4) tion buffer (20 mM Na cacodylate pH 7.0, 5 mM MgCl , 2 R (%) 6.3 (24.0) 6.9 (13.5) 4.2 (10.4) sym 300 mM KCl) and concentrated to 500–600 mM. Phasing power centric 1.0 0.87 – Crystallization was performed in sitting drop by adding acentric (iso/ano) 1.0/– 1.0/– /2.5 one volume of crystallization solution made with MPD R (centric) 0.89 0.84 – cullis (20%) and spermine (50 mM) to nine volumes of RNA ˚ ˚ ˚ in the crystallization buffer. Drops were equilibrated at Space group: P2 2 2; unit cell a = 44.0 A, b = 47.5 A, c = 57.7 A. Mean 1 1 figure of merit before/after solvent flattening: 0.38 (0.07)/0.86 (0.80). 37 C with a reservoir made with 50% MPD, 50 mM Na Values in parenthesis are those for outermost shell. cacodylate, 100 mM MgCl and 300 mM KCl. Platelets 2 b Phasing power = <F /LOC>, where LOC is the lack of closure. crystals appeared within 4–10 days and were stabilized at c R =| |F ± F |– F |/|F – F | for centric reflections. cullis PH P H PH P 20 C and cryo-protected by soaking in reservoir solution. Table 2. Data collection summary for divalent metal-soaked Crystal soaking, X-ray data collection and processing structures Heavy atom derivatives for the MIR method were 2+ 2+ 2+ 2+ obtained by soaking crystals for several days in a reservoir Mn Zn Co Ba solution containing 25 mM ruthenium(III) hexamine X-ray source ESRF ESRF Rot. anode Rot. anode (without magnesium) or 10 mM AuCl (in presence of BM30 BM30 100 mM MgCl ). Soaking of crystals was also performed 2 ˚ Wavelength (A) 1.41 1.28 1.54 1.54 for the determination of the binding sites of cations Max. Resolution (A) 2.60 1.90 2.35 2.56 Completeness (%) 98.4 (92.7) 87.0 (41.9) 94.2 (94.6) 99.2 (94.5) inducing cleavage. Daylong soaking of the crystals in 2+ 2+ Redundancy 3.3 4.8 2.4 5.6 100 mM Mn or Ba was possible without affecting Mean I/ 21.1 (12.0) 19.7 (10.1) 14.1 (2.2) 27.5 (12.4) their diffraction power; for zinc and cobalt, it was neces- R (%) 6.0 (8.3) 6.8 (8.5) 6.1 (38.2) 6.5 (13.2) sym sary to reduce salt concentration to 20 mM and the soaking time to 1 h. After stabilization, crystals were Values in parentheses are those for outermost shell. flash-cooled in liquid ethane (simple flash cooling under the nitrogen gas stream usually resulted in a serious loss of diffraction). All data were collected at 100 K (Tables 1 were localized by Fourier-difference maps. Phasing was and 2), and processed using the HKL package (28). initiated at 2.0 A resolution with SHARP 3.0 (31) using the gold derivative, and not the native, as the reference Structure solution and refinement of structures dataset. The figure of merit was greatly improved by Two strong heavy atom sites were localized using anom- solvent flattening with SOLOMON (32) using a solvent alous differences with LOCHVAT (29,30) for the gold content of 40%. The resulting experimental map was of derivative. Subsequently, three ruthenium-binding sites excellent quality, revealing one dimer per asymmetric unit Nucleic Acids Research, 2010, Vol. 38, No. 17 5809 packed along the c axis so as to form pseudo-infinite the results show a very similar pattern at the two pH helices. The model was built with O 6.2 (33) and structures values (Figure 5b) may be viewed as an indication that of pre-cleaved and post-cleaved were refined with CNS the procedure was effective. All calculations, comprising (34) (Table 3). Potassium, zinc, cobalt and manganese image gel processing, were made with Mathematica from ions were identified using anomalous Fourier-difference Wolfram Research. maps as described (19). Analysis of RNA cleavage in solution RESULTS The 23-mer DIS RNA at a concentration of 90 mMin Description of the structure water was heated at 90 C for 5 min. and slowly cooled to room temperature for several hours to allow duplex As observed in previously described crystal structures of formation. The sample was then diluted to 12 mMina DIS extended duplex, the subtype-B DIS extended duplex buffer containing 150 mM potassium acetate, 20 mM structure looks quite similar to its kissing-complex coun- sodium cacodylate pH 6.85 or pH 6.31, and 5 or 20 mM terpart (21,22) in spite of the different RNA topology of divalent cations (MgCl , MnCl , ZnCl , CoCl , BaCl , (Supplementary Figure S2). The structure is mainly 2 2 2 2 2 Pb(II) acetate) or 3 mM EDTA, and incubated at 37 C. characterized by A and A of both strands related 272 273 Lead acetate was used for its higher solubility in compari- by non-crystallographic symmetry (named strands a and son of lead chloride. Slightly acidic conditions prevented b in the following) being extrahelical and forming a the formation of insoluble hydroxides. For visualization four-base stack, the A of each strand remaining of the 5 -cleavage product and quantification, unpaired inside the helix (Figure 2). A comparison of 0 32 5 [g P]ATP-labelled RNA was mixed with unlabelled these flanking purines among various DIS crystal struc- RNA. The RNA was analysed on denaturing 8 M urea, tures are described in Supplementary Data 20% polyacrylamide gel electrophoresis (DPAGE) in TBE (Supplementary Figure S3). In spite of a local phosphate buffer (45 mM tris–borate pH 8.3, 2 mM EDTA) and the backbone distortion induced by the inter-strand adenine cleavage position was checked using RNase T1 and stacking, the duplex remains perfectly linear (Figure 2b). alkaline ladders. Gel quantification was made with a In the present structure, A and A are involved in 273a 273b Fuji phosphoimager. Raw data from the phosphoimager ‘A-minor’ (35,36) contacts with the minor groove of a software were obtained as binary image files. To trans- symmetry-related duplex molecule, leading to an inter- form the raw value at each pixel of the image into a action very similar to one described in the 30 S ribosomal value proportional to the radioactivity count, the follow- subunit [see Figure 7b in (37)]. It may be hypothesized that 4RawValue ing transformation RawValue ! 10 (unim- such an interaction occurs within the complete portant pre-exponential factors are omitted) was initially applied, according to information from the manufacturer. We found it necessary to refine the latter transformation as 3:86RawValue RawValue ! 10 to fit correctly the response obtained from a known dilution scale. When the 2+ cleavage was fast enough, as for Pb , the cleavage rate constants k could have been derived following the relation kt f ¼ 1  e where f is the fraction of cleaved RNA obs obs (Supplementary Figure S13b). However, in situations of 2+ 2+ 2+ slow and incomplete cleavage, as for Co ,Mn or Mg , the inescapable variations in the amount of radioactive materials loaded in each lane of the gel made this proced- ure too inaccurate. An internal standardization was thus used within each lane by considering also the small-size products (Supplementary Figures S10 and S13a). The pro- cedure is explained in Supplementary Data. The fact that Table 3. Refinement statistics 2+ 2+ Native Mn soaked Co soaked Max. resolution (A) 1.6 2.6 2.4 Completeness (work + test sets) 95.4% 97.7% 91.2% R factor 25.0% 23.0% 28.4% free R factor 24.2% 20.0% 23.9% Estimated coordinate errors (A) 0.22 0.34 0.36 Average B factor (A ) 32.0 20.4 42.0 Figure 2. Structure of the HIV-1 subtype-B DIS extended duplex. The Number of nucleic acid atoms 982 982 982 two strands are represented in green and red. The black arrowhead Number of water molecules 183 149 46 depicts the asymmetric cleavage site observed in crystals. (a) Number of metal atoms 5 8 5 Sequence and secondary structure of the duplex. (b) Stereo view of Protein Data Bank ID 2OIY 2OJ0 3FAR the structure solved in this study. 5810 Nucleic Acids Research, 2010, Vol. 38, No. 17 encapsidation region in the viral RNA after dimerization conformations of G ), no significant cleavage could be 271a and maturation of virions (38,39). detected at G on the other strand related by 271b non-crystallographic symmetry. In agreement with this The subtype-B DIS duplex is cleaved in crystals and in observation the ribose of G clearly appeared in the 271b solution C2 -endo conformation as predominantly observed in absence of cleavage at that stem terminal base-pair The most striking feature of the subtype-B duplex struc- (21,22). Altogether, these observations are strongly in ture is the presence of a cleavage between G and A of 271 272 favour of the existence of cation-dependent cleavage in one of the two strands (strand a in green on Figure 2b). the crystals. This obviously does not exclude that a part The cleavage was first revealed during refinement of the of the RNA molecules integrated in a crystal were previ- structure by inspection of electron density maps from ously cleaved in solution. several crystals. It appeared on (Fo–Fc) difference Fourier maps as a strong positive peak close to ribose Crystallographic study of cation binding near 271a, which corresponds to the position of the phosphate the cleavage site after cleavage, and as a strong negative peak on its expected position before cleavage (Figure 3a). In agree- To understand the role of metal ions in the primary ment with this interpretation, a composite simulated an- cleavage mechanism, we soaked crystals for several days nealing omit map showed a very poor density for the with magnesium-free solutions containing either manga- ribose of G in a C2 -endo conformation, and for the nese, zinc, barium or cobalt divalent cations. Anomalous 271a G -A phosphodiester bond. The cleavage was con- difference maps for manganese, zinc and cobalt (for 271a 272a firmed by a polyacrylamide gel analysis of RNA from barium, see below) revealed a strong peak in the vicinity dissolved crystals and from drops where they had grown of G for manganese [11.5 estimated standard devi- 271a (Figure 3b). Furthermore, inspection of various electron ations (e.s.d.)], zinc (19.5 e.s.d.) and cobalt (8.9 e.s.d.) density maps clearly showed an alternative C3 -endo con- (Figure 4a and Supplementary Figures S5 and S6). In formation of the G ribose and a 3 -phosphate resulting addition, simulated annealing composite omit maps 271a from cleavage. This suggested that cleavage proceeds showed a conformational change of the sugar–phosphate through the classical mechanism of nucleolytic ribozymes backbone between residues 273a and 274a, resulting in an by a two-step reaction involving first a trans-esterification inward rotation of phosphate 274a and an outward 2.3 A 0 0 producing strand scission and a 2 -3 -cyclic phosphate translation movement of G (Figure 4b). This local con- 274a intermediate and, second, followed by its hydrolysis re- formational change creates a negatively charged metal ion sulting in the 3 -phosphate product [Supplementary pocket, where the partially dehydrated divalent ion is Figure S4; for review, see (40)]. Such a mechanism was directly bound to N7 of G , and to anionic oxygen 274a first observed for the tRNA(Phe) (41,42), and for atoms of phosphates 273a and 274a (Figure 4a). This con- lead-dependent ribozymes (43), but not with hammerhead, trasts with the catalytic divalent cation bound by hairpin and hepatitis d ribozymes that are unable to outer-sphere coordination to the active site of the HDV 0 0 catalyse hydrolysis of the 2 -3 -cyclic phosphate ribozyme (46). Notably, the residues surrounding G , 274a produced by the first trans-esterification [for review, see including the bulged adenines, are not affected by this (44,45)]. motion, and the base pair G -C is not disrupted 274a 279b Surprisingly, whereas one strand in the crystals was (Figure 4a). Although the cation binding scheme is 2+ 2+ cleaved to within 30–40% (which was estimated by similar for Mn and Zn , the distance from each refining the occupancy of the pre- and post-cleaved cation to the 2 -OH group of the cleaved G residue 271a Figure 3. Cleavage observed in the HIV-1 subtype-B extended duplex. (a) Stereo view of the cleaved region. The model used for electron density map calculations corresponds to the uncleaved form. (3Fo–2Fc) electron density map contoured at 1.4 e.s.d. is shown in blue; (Fo–Fc) difference map is shown contoured at 5.0 e.s.d. (red), and –3.5 e.s.d. (orange). The latter shows an excess of density on phosphate 272 as built in the uncleaved model (negative peak), and a lack of density (positive peak) corresponding to its position after the cleavage. (b) Denaturing PAGE analysis of RNA extracted from crystallization drop, from a dissolved crystal, and before crystallization (0). Only the largest 5 -fragment resulting from cleavage is visible upon ethidium bromide staining. Nucleic Acids Research, 2010, Vol. 38, No. 17 5811 Figure 4. Cation-induced conformational change at the A –G step. (a) Stereo view of the composite simulated annealing omit map contoured at 273 274 1.4 e.s.d. showing a clear density for residues A and G after the conformation change (indicated with black arrows), without disrupting the 273a 274a G –C base-pair. The anomalous-difference map corresponding to the manganese ion is represented in orange and contoured at 8.0 e.s.d. The 274a 281b ˚ ˚ ˚ cation lies at 2.4 A from the N7(G ) and at 2.3 A and 2.0 A from each phosphate. (b) Superimposition of the native (in gray) and the 274a 2+ 2+ 2+ manganese-soaked structures (in blue). The binding of Mn ,Co or Zn induces a 2.3 A movement of base and ribose 274, as well as a rotation of phosphate 273 toward the interior of the helix (pink circled region). 2+ 2+ ˚ ˚ varies from 6.5 A for Mn to 5.6 A for Zn . For the Two strong peaks in an anomalous difference map (13.2 2+ latter cation, the anomalous difference map displays a and 11.5 e.s.d.) were also seen with Ba on G and 274a G (Supplementary Figure S7). At variance with other major peak and also, well above the background, an 274b 2+ cations, Ba is shifted toward O6 of G and G , elongated extension which may be the mark of a more 274a 274b which prevents it from a direct coordination with phos- complex binding mode (Supplementary Figure S5). phates 273 and 274. Also, no cleavage was visible with Interestingly, no significant peak was detected in anomal- 2+ 2+ 2+ Ba at G and some cleavage was visible at G , 271b 271a ous difference map for Zn and Mn on G on the 274b but very likely this cleavage was due to the presence of other strand and, concomitantly, the electron density map 2+ 2+ Mg prior to soaking with Ba . Such a peculiarity did not reveal any cleavage at G . 271b 2+ will be rationalized fully in the following. Finally, this For Co , on the contrary, a strong peak (7.4 e.s.d.) was cation binding study also showed, as previously reported also found in an anomalous difference map on G and, 274b (27), that metal ion binding to RNA can be very sensitive concomitantly, a cleavage at G was observed 271b 2+ 2+ to small structural variations since only Ba and Co (Supplementary Figure S6). This confirms that cleavage were able to bind at the two a priori equivalent sites can take place in the crystals and that divalent cations 2+ G and G . Such a non-symmetric binding for 274a 274b are involved in the cleavage. The binding mode of Co 2+ 2+ 2+ 2+ Mn and Zn is likely limited to the crystalline state also differs from Zn and Mn because, first, no inward and, therefore, both strands should be susceptible to movement of phosphate 274 is observed, which results in a cleavage in solution. direct coordination of the cation only to N7 of G and, 2+ 0 second, because the distances between Co and 2 -OH Solution study of the cleavage groups of G and G (4.8 and 3.8 A) are shorter 274a 274b 2+ than for their Zn counterparts (Supplementary Figure Although the involvement of the G 2 -OH group in the S6). It is difficult to decide whether these differences are cleavage reaction was extremely probable, we confirmed it related to the different nature of each cation, to enhanced by using a mutated DIS sequence where this residue was cleavage in the crystal, or to both. replaced by a deoxyguanosine; as expected, cleaving 5812 Nucleic Acids Research, 2010, Vol. 38, No. 17 activity was abolished (Supplementary Figure S8). We correlates very well with their shifting of the pKa of 2+ 2+ 2+ 2+ then compared the ability of Mg ,Mn ,Zn ,Co , coordinated water molecules since we obtained 2+ 2+ 2+ Pb ,Ba and Ca to induce strand cleavage in  log k=pKa ¼1:06  0:1 at pH 6.30 and solution. Experiments were performed at 37 C and result-  log k=pKa ¼0:93  0:14 at pH 6.85, where k is the ing fragments were separated by denaturing PAGE. cleavage rate constant (Figure 5b). Therefore, Ethidium bromide staining revealed the 16-mer fragment  log k=pKa ¼1 within experimental errors. We also corresponding to the 3 cleavage product. This also observed that lowering the pH by 0.55 unit (from 6.85 to revealed a distinct band, which was unexpected since 6.3) resulted in less cleavage for all cations with a mean only one cleavage site could be seen in the crystal structure value  log k=pH ¼ 0:98  0:16 (Figure 5b). Thus, (Figure 5a). We note that this additional band was absent within experimental errors, there exists a pKa/pH com- 2+ with Co . Essentially, this was the mark of an additional pensation on the kinetics of cleavage, which implies that cleavage site at A280, opposite to the cation binding site. a cation coordinated to a hydroxide ion is responsible for More details about this are in Supplementary Data. In the the activation of the 2 -OH group of G leading to 271a following, the cleavage at G271 and at A280 will be cleavage. Indeed, considering the acid-base equilibrium 2+ 2+ referred to, respectively, as the primary and the secondary between the two species M (H O) and M (H O) 2 n 2 n–1 pHpK pHpK a a cleavage cleavage site. OH yields m ¼ m 10 =ð1+10 Þ, where m is the Under conditions close to those used for crystallization concentration of the cation bearing a hydroxide anion and 2+ (20 mM Mg ), the primary cleavage is rather slow m is the total cation concentration. This accounts well for compared to small RNA ribozymes. Only 7% of the the observed pKa/pH compensation. If the active cationic species were not to be bound to the RNA was cleaved after 14 h of incubation (Figure 5), 5 1 which yields a rate constant k 2+= 8.6  10 min .In RNA at some stage prior cleavage, one would expect that, Mg presence of either 3 mM EDTA and 100 mM Na ,or of at a given pH, the rate of cleavage should be proportional saturating concentration of cobalt(III) hexammine, no to the total cation concentration m . However, with man- cleavage was detectable even on daylong time-scale, con- ganese, for which accurate data could be obtained at both firming that divalent metal ions are required for the pH, a 4-fold increase of total concentration from 5 to reaction (Supplementary Figure S9 and not shown). We 20 mM yielded k(20 mM)/k(5 mM) = 2.2 ± 0.15 at have thus compared the efficiency of cleavage with differ- pH 6.85 and k(20 mM)/k(5 mM) = 2.4 ± 0.5 at pH 6.30, 2+ 2+ 2+ 2+ 2+ 2+ ent cations: Ca ,Ba ,Mn ,Co ,Zn and Pb which are values significantly different from 4. Such a (Figure 5 and Supplementary Figures S9, S10 and S11). value close to 2.3 agrees well with the need of cation Cleavage rates observed for the DIS duplex are compar- binding to the RNA before cleavage if the binding of 2+ able to those observed for the secondary cleavage sites in Mn at 20 mM is in excess to its binding at 5 mM pre- the minimal hammerhead ribozyme (47,48). In our case, cisely by a factor 2.3. For this to take place the dissoci- 2+ 2+ the most efficient cation is Pb , which led to 42% of ation constant of Mn has to be K = 9 mM. 2+ Interestingly, a much more detailed analysis of the same cleavage after 1 h at 5 mM concentration (k = Pb 2 1 2+ 2+ kind was performed for the binding of magnesium to the 10 min ), and the less efficient are Ca and Ba , which did not promote any detectable cleavage after 14 h HDV ribozyme and led to quite comparable values (Figure 5a and data not shown). (K = 3.1 mM at pH 6.5) (49). Furthermore, our value Analysis of the cleavage rates showed that the ranking K = 9 mM fits also well with apparent dissociation con- of the tested cations for their ability to cleave the duplex stants of various divalent cations (from 5 to 12 mM) that Figure 5. Analysis of the cleavage of 23-mer subtype-B DIS duplex in solution. (a) Metal ion dependency of the cleavage at pH 6.85 and 37 C. The 2+ cleavage with Zn was also performed at pH 6.3. (b) Variation at two pH values of the cleavage rate versus the pKa of each cation at 5 mM. The 2+ kinetic constant could not be determined at the lower pH for Mg . The error bars are those from the numeric procedure exposed in Supplementary Data and are not fully representative of the overall errors which are certainly more important (see text). Nucleic Acids Research, 2010, Vol. 38, No. 17 5813 were reported for a modified Schitosoma hammerhead ribozyme (50). Finally, we also performed competition ex- 2+ 2+ periments with Ba and Ca that are inefficient at 2+ 2+ cleaving the RNA. As expected, Ba as well as Ca in- 2+ hibited the Co -dependent cleavage (Supplementary 2+ Figure S12). This is consistent with Co being dislodged from the specific binding sites by competitive inhibitors (albeit we have a formal crystallographic proof of 2+ specific binding only for Ba ; see Supplementary 2+ Figure S7). Half inhibition by Ba was seen to take place roughly in the range 6–30 mM (Supplementary Figure S12). Calculation of the inhibition factor shows 2+ that this is explained if the K of Ba is comprised between 5 and 25 mM, which seems also reasonable in view of the values just mentioned. Although competitive inhibition agrees with our results, one cannot exclude that, 2+ 2+ at the highest Ba or Ca concentrations, electrostatic screening too (51), and not only direct competition for the crucial binding sites, was responsible for cleavage inhibition. DISCUSSION Proposition of a detailed cleavage mechanism By using the different views of the cleavage site provided by our crystal structures, as well as the results from solution studies, we can propose the following cleavage mechanism for the DIS duplex (Supplementary Figure S4). Clearly, the rather slow cleaving rate observed in this structure compared to ‘standard ribozymes’ is an ad- vantage for crystallographic studies by allowing the trapping of intermediate states. First, a snapshot of the structure before cleavage is provided by the uncleaved strand, which shows ribose 271 in a C2 -endo conform- ation (Figure 6a). Second, there would be a necessary intermediate consisting in the binding of a divalent cation with a suitable pKa at the A –G step; depend- 273 274 ing on the cation, there is a local rearrangement leading to the involvement of A ,G phosphates in the binding 273 274 (Figure 6b). The metal ion would then be able to provide a Figure 6. Proposition for a cleavage mechanism. (a) Snapshot of the hydroxide anion accepting a proton from the 2 -OH group initial state provided by the uncleaved strand. Residue G is in a C2 - of G . Third, the cleavage reaction would require a endo conformation and no metal ion is present in the pocket, which is closed by an unusual CH8(G )-phosphate(A ) hydrogen bond. The 274 273 crankshaft motion of the A phosphate towards the (3Fo–2Fc) electron density map contoured at 1.4 e.s.d. is shown in blue. interior of the helix to obtain a configuration of the A The gray arrowhead shows the cleavable phosphodiester bond. (b)A 0 0 2+ scissile bond in line with the O2 of G in a C2 -endo divalent metal ion (here Mn ) binds at the A –G step, inducing a 273 274 conformation. A model of such a conformation is shown rearrangement of the sugar-phosphate backbone. The anomalous-difference map contoured at 8.0 e.s.d. is shown in orange. in Figure 6c. This unstable phosphate conformation, very (c) The metal ion reduces the pKa of a bound water molecule and likely the chemical limiting step, was not observed in our generates hydroxide anions that activate the 2 -OH group of the G crystal structure, but would be very similar to the one ribose. A necessary movement of the phosphate 272 toward the interior observed at the cleavage site of the fully active extended of the helix (curved arrow #1) then orients the cleavable bond in-line hammerhead ribozyme (52). Such an in-line conformation with the activated 2 -hydroxyl group thus allowing (curved arrow #2) a 0 0 nucleophilic attack leading to a 2 -3 -cyclic phosphate intermediate. was also observed at a secondary cleavage site of the This product is not stable in crystals and is hydrolyzed (d) into a minimal hammerhead ribozyme where it was stabilized 3 -monophosphate (in red) product observed in this simulated anneal- by a direct interaction with a zinc cation (48) ing composite omit map (contoured at 1.4 e.s.d.). (Supplementary Figure S13). Such a 2 -OH-mediated 3 -phosphate product as previously observed with the nucleophilic attack can only result in strand scission 0 0 0 leadzyme (43). In the absence of a cyclic phosphate the with a 2 -3 -cyclic phosphate and 5 -OH termini. Since 0 0 the 2 -3 -cyclic phosphate could not be detected in the G ribose is no more constrained and is free to switch 0 0 electron density map, this means that it should be from its initial C2 -endo to a C3 -endo conformation rec- hydrolyzed quickly enough to yield the observed ognizable in electron density maps (Figure 6d). 5814 Nucleic Acids Research, 2010, Vol. 38, No. 17 Clearly, one conflicting observation should be ad- A Trojan horse mechanism? 2+ dressed. Indeed, there is a difference between Mg , Since cation binding is a prerequisite for cleavage and which was not visible in the electron density map and since the active species corresponds to a partially yet is able to cleave (since the original observation of neutralized cation carrying a hydroxide anion, a doubly 2+ cleavage was in a Mg -containing crystal), and other charged cation should be a strong inhibitor of the cleavage cleaving cations that were visible in electron density for two reasons. First, this is because its concentration is 2+ maps. This is not unprecedented since the catalytic Pb higher than that of the active species; for zinc for example, in the leadzyme ribozyme for example (53) could not be 2+ it can be estimated from (56) that [Zn ] is in 20-fold 2+ localized in crystal structures. In the present case, we have excess versus [Zn OH ]. Secondly, the affinity for the 2+ no clear-cut explanation for this difference between Mg anionic binding site of the doubly charged species is cer- and other cations. Nevertheless, it may be mentioned that tainly also much higher than that of the singly charged 2+ Mg (10 electrons) is inherently more difficult to observe species. It is thus tempting to suggest a scenario in two 2+ than the other cations we used (from 23 to 80 electrons). steps. First, such an inactive doubly charged M species Furthermore, magnesium has an exceedingly small anom- would indeed bind, and well, but without being protected alous scattering component, which makes useless an against the H O/OH exchange. Second, once the cation 2+ anomalous difference map to reveal its presence in a has been partially neutralized as M (OH ), and thus 2+ specific binding site, if any. Finally, Mg might also transformed in situ into the active singly charged species, 2+ 2+ 2+ bind less strongly than Mn ,Co and Zn , but suffi- it would be also more easily released in the immediate 2+ ciently to provoke cleavage, and/or Mg is released after vicinity of the cleavable site. Expressed in a metaphoric 2+ 2+ 2+ cleavage more easily than Mn ,Co and Zn . Either way, the cleavage would result from some ‘Trojan horse’ possibility is consistent with the stronger affinity for N7 mechanism wherein an apparently ‘inoffensive’ cation 2+ atom of the latter cations that are softer than Mg [in this species is hosted next to the cleavage site but, soon or respect, see (54)]. later, is transformed into a more easily released ‘aggres- It is interesting to compare the cleavage pattern sive’ species. The interesting consequence is that the more observed in the DIS RNA after cation binding at a tightly bound an inhibitor is, the more chance it has to be specific site with an artificial system using lanthanide transformed in situ into the aggressive species. Such an complexes covalently linked to an oligoDNA hybridized interpretation should be of general extent in situations to an oligoRNA (55). When a perfect helical duplex where a bound cation is active as the general base (as, was formed, due to full complementarity of the RNA we think, this holds true in the present case). and DNA sequences, a cleavage was observed, Comparison with other cation-dependent ribozyme but only at the phosphodiester bond opposite to the nucleotide bearing the lanthanide complex. Interestingly, This DIS fragment should be viewed as a metal-dependent the position opposite to the cation binding site in the DIS ribozyme, but certainly not as a very efficient one. There is A280 (Figure 2), where a secondary cleavage was are two reasons for that. First, the necessary conform- also observed (Supplementary Figures S10 and S11). ational change yielding the in-line geometry (Figure 6c) When the artificial system was designed with an imperfect requires a high, and may be even a very high, activation sequence complementarity allowing an RNA bulge to energy. Second, we did not detect any candidate to act as form in the vicinity of the lanthanide complex, up the general acid providing a proton to the leaving group. to four additional consecutive cuts were observed in Very likely, this role is played by a water molecule from the stretch of unpaired residues forming the bulge. the bulk solvent. On the contrary, in very efficient ribo- Overall, the latter observation is strikingly similar zymes, the structure is tuned for a favourable orientation to what we observed with the DIS, the primary cleavage of the cleavable bond and, furthermore, both the general site being at G271 next to the extrahelical purines A272, base and the general acid are identified as pre-structured A273 corresponding to the bulged-out residues in entities. This is the case, for example, for the hepatitis the artificial system. However, in our case, a more delta virus (HDV) ribozyme where a bound magnesium specific pattern was seen with cobalt which produced no cation and a cytosine base (C75) were clearly identified. significant cleavage at A280 (opposite to the cation However, somewhat strangely, no definite consensus binding site) and almost no additional cleavage surround- emerged about which acts as the general acid, and which ing the major cleavage site at G271 (Supplementary acts as the general base (46,57–59). A ‘multichannel Figure S11). This is certainly related, albeit not in an reaction mechanism’, instead of a presupposed unique obvious way, to the previously mentioned different mechanism, could reconcile the opposite views (49). binding mode of cobalt versus zinc and manganese. Recently, a new metal ion binding site was described at Therefore, what this comparison shows is that the gross a position that is consistent with a magnesium-bound hy- features of the cleavage pattern in the DIS do not seem droxide serving as a general base (60). This would there- to derive from the genuine specificity of binding of fore be comparable to our observations. divalent cations, but rather from the cation binding site In the case of the DIS ribozyme, although the role of the being close to the covalent bond susceptible to cleavage cation as the general base seems established, it is unclear (see the ‘Conclusion’ section). Some peculiarities, whether the hydroxide anion has to be shuttled by the however, do result from the specificity of binding, as cation to the accepting hydroxyl group, or whether it seen with cobalt. may simply be released by the cation close to it. In the Nucleic Acids Research, 2010, Vol. 38, No. 17 5815 6. Paillart,J.-C., Berthoux,L., Ottmann,M., Darlix,J.-L., Marquet,R., first case, the cation would be directly involved into the Ehresmann,C. and Ehresmann,B. (1996) A dual role of the chemical step (possibly interacting also with the A272 dimerization initiation site of HIV-1 in genomic RNA packaging phosphate after its conformational change (Figure 6c), and proviral DNA synthesis. J. Virol., 70, 8348–8354. which would stabilize the transition state), whereas in 7. Berkhout,B. and van Wamel,J.L. (1996) Role of the DIS hairpin the second case it would act at a distance. In this in replication of human immunodeficiency virus type 1. J. Virol., 70, 6723–6732. respect, a thorough comparison with protein enzymes 8. Clever,J.L. and Parslow,T.G. (1997) Mutant HIV-1 genomes with has led to the proposal that a cation can indeed exert a defects in RNA dimerization or encapsidation. J. Virol., 71, catalytic effect without contacting directly its ‘target’ (61). 3407–3414. This is certainly not against the previous conclusion drawn 9. Laughrea,M., Jette´ ,L., Mak,J., Kleinman,L., Liang,C. and Wainberg,M.A. (1997) Mutations in the kissing-loop hairpin of from the comparison with the artificial system using lan- human immunodeficiency virus type 1 reduce viral infectivity as thanide complexes (55). well as genomic RNA packaging and dimerization. J. Virol., 71, 3397–3406. 10. Laughrea,M. and Jette´ ,L. (1996) Kissing-loop model of HIV-1 ACCESSION NUMBERS genome dimerization: HIV-1 RNA can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin Atomic coordinates and structure factors of the native, the 248-271 are dispensable for dimer formation. Biochemistry, 35, manganese- and the cobalt-soaked structures have been 1589–1598. 11. Muriaux,D., Fosse´ ,P. and Paoletti,J. (1996) A kissing complex deposited with the Protein Data Bank (ID 2OIY, 2OJ0 together with a stable dimer is involved in the HIV-1 RNA Lai and 3FAR, respectively). dimerization process in vitro. Biochemistry, 35, 5075–5082. 12. Muriaux,D., Rocquigny,H.D., Roques,B.P. and Paoletti,J. (1996) NCP7 activates HIV-1 Lai RNA dimerization by converting a SUPPLEMENTARY DATA transient loop-loop complex into a stable dimer. J. Biol. Chem., 271, 33686–33692. Supplementary Data are available at NAR Online. 13. Takahashi,K.I., Baba,S., Chattopadhyay,P., Koyanagi,Y., Yamamoto,N., Takaku,H. and Kawai,G. (2000) Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome. RNA, 6, 96–102. ACKNOWLEDGEMENTS 14. Takahashi,K.I., Baba,S., Koyanagi,Y., Yamamoto,N., Takaku,H. The authors thank Philippe Carpentier for his abundant and Kawai,G. 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Published: Sep 11, 2010

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