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. 2013 Dec;19(12):1703-10.
doi: 10.1261/rna.041517.113. Epub 2013 Oct 22.

The molecular recognition of kink-turn structure by the L7Ae class of proteins

Lin Huang, David M J Lilley

The molecular recognition of kink-turn structure by the L7Ae class of proteins

Lin Huang et al. RNA. 2013 Dec.

Abstract

L7Ae is a member of a protein family that binds kink-turns (k-turns) in many functional RNA species. We have solved the X-ray crystal structure of the near-consensus sequence Kt-7 of Haloarcula marismortui bound by Archaeoglobus fulgidus L7Ae at 2.3-Å resolution. We also present a structure of Kt-7 in the absence of bound protein at 2.2-Å resolution. As a result, we can describe a general mode of recognition of k-turn structure by the L7Ae family proteins. The protein makes interactions in the widened major groove on the outer face of the k-turn. Two regions of the protein are involved. One is an α-helix that enters the major groove of the NC helix, making both nonspecific backbone interactions and specific interactions with the guanine nucleobases of the conserved G • A pairs. A hydrophobic loop makes close contact with the L1 and L2 bases, and a glutamate side chain hydrogen bonds with L1. Taken together, these interactions are highly selective for the structure of the k-turn and suggest how conformational selection of the folded k-turn occurs.

Keywords: RNA structure; RNA-protein recognition; X-ray crystallography; k-turn motif.

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Figures

FIGURE 1.
FIGURE 1.
Sequence of Kt-7 and the RNA species used for crystallization. (A) The sequence of H. marismortui Kt-7, with key nucleotides labeled using our standard nomenclature (Liu and Lilley 2007). (B) A stem–loop RNA containing a single Kt-7 sequence. This was used for the crystallization of the complex of A. fulgidus L7Ae bound to Kt-7. (C) A duplex RNA containing two Kt-7 sequences with twofold rotational symmetry. This was crystallized in the absence of protein.
FIGURE 2.
FIGURE 2.
The structure of the complex of L7Ae bound to Kt-7. Stereoscopic pairs (A,D,E) are shown as parallel-eye stereoscopic pairs. Electron density is taken from the composite omit map and contoured at 2σ. (A) An overall view of the complex. The basic β-strand:turn:helix and hydrophobic loop sections are colored blue, with hydrogen bonds between amino acid side chains and the RNA highlighted in red. The nucleotides of the k-turn are colored in our conventional scheme (loop purple; G•A pairs green; NC helix gray; C helix yellow), with the NC and C helices indicated. (B,C) Nonstereoscopic images of the structures of the two recognition elements of the protein, i.e., the α-helix (B) and the hydrophobic loop (C). (D,E) The interaction between the α-helix (D) and the hydrophobic loop (E) of the k-turn RNA. The electron density map is shown for the protein. Hydrogen bonds between amino acid side chains and the RNA are highlighted in red. Note that the N-terminal end of the α-helix is directed toward O6 of G1b.
FIGURE 3.
FIGURE 3.
The structure of Kt-7 in the complex with L7Ae and in the absence of bound protein. These are shown as parallel-eye stereoscopic pairs. (A) The complete k-turn structure, viewed from the side of the bulged strand, with the electron density shown contoured at 2σ is taken from the composite omit map. (B,C) The structures of Kt-7 in the complex (B) and protein-free (C), both viewed from the nonbulged strand side with the C-helix directed rightward.
FIGURE 4.
FIGURE 4.
Superposition of crystal structures of Kt-7. The structure of Kt-7 from the complex with L7Ae (PDB:4BW0) is shown in our standard coloring. The structures are viewed from the side of the bulged strand. The images are shown in parallel-eye stereo. (A) Superimposition with the structure of Kt-7 as a protein-free duplex colored cyan (PDB:4C40). The RMSD between the two structures is 0.83 Å. (B) Superimposition with the structure of Kt-7 inserted into the SAM-I riboswitch (Daldrop and Lilley 2013) colored orange (PDB:4B5R). The RMSD between the two structures is 1.13 Å. Note that all three Kt-7 structures are in the N3 conformation.
FIGURE 5.
FIGURE 5.
Comparison of the G2n•A2b–A-1n interaction in four Kt-7 structures. (A) Bound by L7Ae protein (this work, with electron density from the composite omit map); (B) as free RNA (this work, with electron density from the composite omit map); (C) engineered into the SAM-I riboswitch (Daldrop and Lilley 2013); and (D) in the H. marismortui ribosomal 50S subunit 23S rRNA (Ban et al. 2000). Structures A–C fall into the N3 class, with G-1n O2′ donating a proton to A2b N3 and two hydrogen bonds connecting A2b and G2n, whereas the ribosomal structure (D) is N1 class where G-1n O2′ donates a proton to A2b N1. The resulting rotation of the A2b nucleobase results in an A2b N6 to G2n N3 N–N length of 4.3 Å, i.e., too long to be hydrogen bonded.
FIGURE 6.
FIGURE 6.
Scheme showing two alternative pathways of k-turn folding upon binding to L7Ae protein. The Kt-7 RNA is in equilibrium between the extended and folded conformation in free solution, biased toward the extended state at low ionic concentration. In the conformational selection mechanism (the vertical equilibrium shown right), the L7Ae selectively binds to the folded form and thus draws the equilibrium toward this state. Alternatively, the protein could bind the extended state and mechanically force the RNA to adopt the k-turn structure in an induced fit process (left). The conformational selection model requires that L7Ae structure is complementary to that of the folded k-turn, and the structure of the k-turn in the absence of protein is closely similar to that of the bound RNA. The structures determined here (shown far right) show that these requirements are met.
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References

    1. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. 2010. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallog 66: 213–221. - PMC - PubMed
    1. Baird NJ, Zhang J, Hamma T, Ferré-D'Amaré AR. 2012. YbxF and YlxQ are bacterial homologs of L7Ae, and bind K-turns but not K-loops. RNA 18: 759–770. - PMC - PubMed
    1. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. 2000. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905–920. - PubMed
    1. Beaucage SL, Caruthers MH. 1981. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett 22: 1859–1862.
    1. Chen VB, Arendall WB III, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Cryst D 66: 12–21. - PMC - PubMed

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