Metal-Binding Sites in the Major Groove of a Large Ribozyme Domain Jamie H Cate and Jennifer a Doudna*

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Researchprovided Article by Elsevier1221 - Publisher Connector Metal-binding sites in the major groove of a large ribozyme domain Jamie H Cate and Jennifer A Doudna* Background: Group I self-splicing introns catalyze sequential Address: Deptartment of Molecular Biophysics transesterification reactions within an RNA transcript to produce the correctly and Biochemistry, Yale University, New Haven, CT spliced product. Often several hundred nucleotides in size, these ribozymes fold 06520, USA. into specific three-dimensional structures that confer activity. The 2.8 Å crystal *Corresponding author. structure of a central component of the Tetrahymena thermophila group I intron, E-mail: [email protected] the 160-nucleotide P4–P6 domain, provides the first detailed view of metal Key words: binding in an RNA large enough to exhibit side-by-side helical packing. The group I intron, MAD phasing, osmium hexammine, RNA long-range contacts and bound ligands that stabilize this fold can now be examined in detail. Received: 5 August 1996 Revisions requested: 29 August 1996 Results: Heavy-atom derivatives used for the structure determination reveal Revisions received: 5 September 1996 Accepted: 5 September 1996 characteristics of some of the metal-binding sites in the P4–P6 domain. Although long-range RNA–RNA contacts within the molecule primarily involve Structure 15 October 1996, 4:1221–1229 the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by © Current Biology Ltd ISSN 0969-2126 G⋅U wobble base pairs. In the native RNA, two of the sites are occupied by fully- hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain. Conclusions: Bound at specific sites in the P4–P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G⋅U base pairs in ribosomal RNAs are magnesium-binding sites. Introduction The 2.8Å crystal structure of the P4–P6 domain [12] pro- Like protein enzymes, catalytic RNAs probably use a vides the first detailed view of an RNA large enough to defined set of structural motifs to build the complex archi- reveal structural motifs directly involved in higher order tectures necessary for function. Divalent metal ions are RNA folding. In the domain, two sets of stacked helices lie essential components of RNA tertiary structure and, in the parallel to one another, connected by a sharp bend at one case of large ribozymes, are required for both stability and end (Fig. 2). Two key interactions stabilize the overall fold catalytic activity [1–6]. A key to understanding and engi- of the molecule. An adenosine-rich bulge docks in the neering ribozyme function lies in the determination of how minor groove of the P4 helix and a GAAA tetraloop binds in metals are bound specifically by RNA and positioned for a the minor groove of its receptor. In addition to base-specific variety of uses. hydrogen bonding and base stacking, these long-range contacts are further secured by pairs of interdigitated riboses In the well studied group I self-splicing introns, magne- termed ribose zippers [12]. sium-dependent folding of the RNA was first detected using Fe(II)-EDTA to probe the accessibility of the phos- Most of the contacts that stabilize internal domain struc- phodiester backbone in solution [4,7]. This experimental ture, as well as the packing of arrays of molecules in the approach revealed that half of the conserved catalytic core crystal lattice, involve the minor groove [12,13]: the wide of the Tetrahymena thermophila intron is part of an indepen- and shallow minor grooves of A-form helices are generally dently folding domain consisting of the base-paired (P) more accessible than the deep major grooves [14]. regions P4 to P6 (P4–P6; Fig. 1) [8]. The P4–P6 domain, However, non-canonical base pairings, loops and bulges in modeled as part of the active site by Michel and Westhof RNA can perturb the geometry of a helix, affecting the [9], folds rapidly in solution [10] and assembles in trans shape and electrostatic potential in localized regions with the other half of the ribozyme to form a functional [15,16]. Here, we show that such local perturbations within complex [11]. the P4–P6 domain give rise to specific metal-binding 1222 Structure 1996, Vol 4 No 10 Figure 1 binding sites expand the known repertoire of ligand- binding motifs in RNA and may provide a general means of derivatizing appropriately designed RNAs for x-ray crys- tallographic analysis. Phylogenetic sequence comparisons show that one class of these motifs occurs frequently in ribosomal RNAs [17], suggesting a mechanism for metal binding in the ribosome. Results and discussion Crystallization conditions suggest a rationale for heavy- atom derivatives The original optimized crystallization conditions for the P4–P6 domain [18] did not produce crystals reproducibly (on average, one crystal per 15 vapor diffusion drops in 1–2 months). To generate the large number of crystals necessary to find heavy-atom derivatives, we developed a pro- tocol using microseeding and cobalt (III) hexammine chloride. When included in crystallization solutions, low concentrations of cobalt hexammine chloride (0.1–1.0mM) dramatically increased the number, size and growth rate of P4–P6 crystals. Crystallization drops pre-equilibrated with cobalt hexammine were microseeded with finely crushed P4–P6 crystals, consistently yielding 2–3 usable crystals per crystallization drop within 2–3 weeks. The diffraction prop- erties of crystals obtained by this procedure were compara- ble with those of crystals obtained by the earlier method. The dramatic effects of cobalt hexammine on crystal growth occurred in spite of a large excess of magnesium ions (25–50mM) in the crystallization conditions, suggesting that specific binding sites for hexammines might be present in the RNA. To take advantage of potential sites, crystals were soaked in stabilizing solutions containing the chemically-related osmium (III) hexammine ion [19]. Initial difference Patterson maps revealed four osmium sites in the asymmetric unit; four additional sites were found by difference Fourier syntheses. In parallel with these experiments, we systematically 3+ 3+ Osmium- and samarium-binding sites in the P4–P6 RNA. Secondary tested metals in the lanthanide series (from Ce to Lu ) structure of the P4–P6 domain; in red, samarium-binding site (Sm); in as potential derivatives, as lanthanides proved useful in the blue, osmium-binding sites. The sites in P5, P5b and P5c bind osmium determination of the tRNA and hammerhead RNA crystal hexammine ions, whereas that in J6/6a binds a putative osmium structures [20–22]. Diffraction experiments on lanthanide- pentammine ion. soaked crystals led us to focus on samarium chloride as a potential heavy-atom derivative for two reasons. First, the pockets in the major groove and near a helical junction. unit-cell dimensions changed as a function of increasing Osmium (III) hexammine, the metal ion used to deter- ionic radius for lanthanides in the series from Lu3+ to Sm3+, mine the RNA structure by multiwavelength anomalous after which they remained constant (Sm3+–Ce3+). Second, diffraction (MAD) phasing, binds at three locations in the the mosaic spread of the diffraction pattern increased as a major groove where non-standard base pairs create pockets function of increasing ionic radius for all lanthanides of negative electrostatic potential. A putative osmium pen- except for Sm3+, for which the mosaicity was only slightly tammine ion binds to a fourth site in the major groove. worse than that of native crystals (0.7° versus 0.5°–0.6°). A Samarium binds between phosphate oxygens in an adeno- full anomalous data set from a samarium-derivatized crystal sine-rich corkscrew structure at the junction of three yielded two strong peaks in an anomalous difference Pat- helices. In three cases, the heavy atoms occupy sites nor- terson map. Samarium-soaked and Sm3+/Os (III) hexam- mally bound by magnesium in the native RNA. The mine double-soaked crystals, compared using difference Research Article Major groove ligands in RNA Cate and Doudna 1223 Figure 2 Locations of metal-binding sites in the crystal structure of the P4–P6 domain. Stereo representation of the crystal structure of P4–P6, rotated 180° from the view in Figure 1 (with the samarium-binding site in red and osmium-binding sites in blue). The figure was produced using the program RIBBONS [51]. Fourier methods, confirmed the osmium sites and pro- from lanthanide binding to the RNA. Biochemical evi- vided the first clear indications of a solvent boundary in dence supports the importance of this region for the overall electron-density maps. Due to poor isomorphism and weak folding of the domain, as well as for ribozyme activity diffraction (to 4.5Å), the samarium derivative was aban- [26,27]. doned in favor of the osmium hexammine derivative. Osmium hexammine binds in the major groove The osmium derivative ultimately provided all of the Osmium hexammine binds at three sites in each P4–P6 phasing power necessary to solve the structure of the P4–P6 molecule in the asymmetric unit. Unlike lanthanides, hexa- domain. Anomalous data from an osmium hexammine- mmines tend to replace weakly bound magnesium ions soaked crystal were measured at two wavelengths near the defined by outer-sphere coordination of the metal [25].

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