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A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

Nature Structural & Molecular Biology volume 18, pages 100–106 (2011)Cite this article

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Abstract

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab–ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs′ high molecular weight, large surface area and phasing power.

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Figure 1: Selection of class I ligase–binding Fabs from the YSGR Fab superlibrary.
Figure 2: Affinity maturation of class I ligase–binding Fab-BL3 by error-prone PCR.
Figure 3: Crystal structure of the Fab BL3-6–ligase complex.
Figure 4: Details of Fab-P5 loop interactions.
Figure 5: Fab–ligase crystal packing.
Figure 6: Analysis of the Fab–ligase P5 epitope.

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References

  1. Eddy, S.R. Noncoding RNA genes. Curr. Opin. Genet. Dev. 9, 695–699 (1999).

    Article  CAS  Google Scholar 

  2. Taft, R.J., Pheasant, M. & Mattick, J.S. The relationship between non-protein-coding DNA and eukaryotic complexity. Bioessays 29, 288–299 (2007).

    Article  CAS  Google Scholar 

  3. Mattick, J.S. The genetic signatures of noncoding RNAs. PLoS Genet. 5, e1000459 (2009).

    Article  Google Scholar 

  4. Cooper, T.A., Wan, L. & Dreyfuss, G. RNA and disease. Cell 136, 777–793 (2009).

    Article  CAS  Google Scholar 

  5. Hogg, J.R. & Collins, K. Structured non-coding RNAs and the RNP Renaissance. Curr. Opin. Chem. Biol. 12, 684–689 (2008).

    Article  CAS  Google Scholar 

  6. Kapranov, P. et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316, 1484–1488 (2007).

    Article  CAS  Google Scholar 

  7. Mattick, J.S. & Makunin, I.V. Non-coding RNA. Hum. Mol. Genet. 15 (Spec No 1), R17–R29 (2006).

    Article  CAS  Google Scholar 

  8. Montange, R.K. & Batey, R.T. Riboswitches: emerging themes in RNA structure and function. Annu Rev Biophys 37, 117–133 (2008).

    Article  CAS  Google Scholar 

  9. Serganov, A. & Patel, D.J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776–790 (2007).

    Article  CAS  Google Scholar 

  10. Fedor, M.J. Comparative enzymology and structural biology of RNA self-cleavage. Ann. Rev. Biophys. 38, 271–299 (2009).

    Article  CAS  Google Scholar 

  11. Hoogstraten, C.G. & Sumita, M. Structure-function relationships in RNA and RNP enzymes: recent advances. Biopolymers 87, 317–328 (2007).

    Article  CAS  Google Scholar 

  12. Jinek, M. & Doudna, J.A. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009).

    Article  CAS  Google Scholar 

  13. Reichow, S.L., Hamma, T., Ferré-D'Amaré, A.R. & Varani, G. The structure and function of small nucleolar ribonucleoproteins. Nucleic Acids Res. 35, 1452–1464 (2007).

    Article  CAS  Google Scholar 

  14. Doudna, J.A. & Cate, J.H. RNA structure: crystal clear? Curr. Opin. Struct. Biol. 7, 310–316 (1997).

    Article  CAS  Google Scholar 

  15. Golden, B.L. & Kundrot, C.E. RNA crystallization. J. Struct. Biol. 142, 98–107 (2003).

    Article  CAS  Google Scholar 

  16. Edwards, A.L., Garst, A.D. & Batey, R.T. Determining structures of RNA aptamers and riboswitches by X-ray crystallography. Methods Mol. Biol. 535, 135–163 (2009).

    Article  CAS  Google Scholar 

  17. Ke, A. & Doudna, J.A. Crystallization of RNA and RNA-protein complexes. Methods 34, 408–414 (2004).

    Article  CAS  Google Scholar 

  18. Ferré-D'Amaré, A.R., Zhou, K. & Doudna, J.A. A general module for RNA crystallization. J. Mol. Biol. 279, 621–631 (1998).

    Article  Google Scholar 

  19. Carrasco, N., Buzin, Y., Tyson, E., Halpert, E. & Huang, Z. Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion. Nucleic Acids Res. 32, 1638–1646 (2004).

    Article  CAS  Google Scholar 

  20. Höbartner, C. et al. Syntheses of RNAs with up to 100 nucleotides containing site-specific 2′-methylseleno labels for use in X-ray crystallography. J. Am. Chem. Soc. 127, 12035–12045 (2005).

    Article  Google Scholar 

  21. Keel, A.Y., Rambo, R.P., Batey, R.T. & Kieft, J.S. A general strategy to solve the phase problem in RNA crystallography. Structure 15, 761–772 (2007).

    Article  CAS  Google Scholar 

  22. Robertson, M.P. & Scott, W.G. A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives. Acta Crystallogr. D Biol. Crystallogr. D64, 738–744 (2008).

    Article  Google Scholar 

  23. Ferré-D'Amaré, A.R. & Doudna, J.A. Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. J. Mol. Biol. 295, 541–556 (2000).

    Article  Google Scholar 

  24. Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376, 660–669 (2002).

    Article  Google Scholar 

  25. Koide, S. Engineering of recombinant crystallization chaperones. Curr. Opin. Struct. Biol. 19, 449–457 (2009).

    Article  CAS  Google Scholar 

  26. Uysal, S. et al. Crystal structure of full-length KcsA in its closed conformation. Proc. Natl. Acad. Sci. USA 106, 6644–6649 (2009).

    Article  CAS  Google Scholar 

  27. Dutzler, R., Campbell, E.B., Cadene, M., Chait, B.T. & MacKinnon, R. X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity. Nature 415, 287–294 (2002).

    Article  CAS  Google Scholar 

  28. Tereshko, V. et al. Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold. Protein Sci. 17, 1175–1187 (2008).

    Article  CAS  Google Scholar 

  29. Koide, S. Engineering of recombinant crystallization chaperones. Curr. Opin. Struct. Biol. 19, 449–457 (2009).

    Article  CAS  Google Scholar 

  30. Carter, P.J. Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343–357 (2006).

    Article  CAS  Google Scholar 

  31. Birtalan, S. et al. The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies. J. Mol. Biol. 377, 1518–1528 (2008).

    Article  CAS  Google Scholar 

  32. Sidhu, S.S., Lowman, H.B., Cunningham, B.C. & Wells, J.A. Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363 (2000).

    Article  CAS  Google Scholar 

  33. Laird-Offringa, I.A. & Belasco, J.G. In vitro genetic analysis of RNA-binding proteins using phage display libraries. Methods Enzymol. 267, 149–168 (1996).

    Article  CAS  Google Scholar 

  34. Ye, J.-D. Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc. Natl. Acad. Sci. USA 105, 82–87 (2008).

    Article  CAS  Google Scholar 

  35. Ekland, E.H., Szostak, J.W. & Bartel, D.P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269, 364–370 (1995).

    Article  CAS  Google Scholar 

  36. Bagby, S.C., Bergman, N.H., Shechner, D.M., Yen, C. & Bartel, D.P. A class I ligase ribozyme with reduced Mg2+ dependence: selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129–2146 (2009).

    Article  CAS  Google Scholar 

  37. Fellouse, F.A., Wiesmann, C. & Sidhu, S.S. Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–12472 (2004).

    Article  CAS  Google Scholar 

  38. Fellouse, F.A. et al. Molecular recognition by a binary code. J. Mol. Biol. 348, 1153 (2005).

    Article  CAS  Google Scholar 

  39. Decanniere, K. et al. A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. Structure 7, 361–370 (1999).

    Article  CAS  Google Scholar 

  40. Desmyter, A. et al. Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J. Biol. Chem. 277, 23645–23650 (2002).

    Article  CAS  Google Scholar 

  41. Shechner, D.M. et al. Crystal structure of the catalytic core of an RNA polymerase ribozyme. Science 326, 1271–1275 (2009).

    Article  CAS  Google Scholar 

  42. Eigenbrot, C., Randal, M., Presta, L., Carter, P. & Kossiakoff, A.A. X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J. Mol. Biol. 229, 969–995 (1993).

    Article  CAS  Google Scholar 

  43. Kossiakoff, A.A. & Koide, S. Understanding mechanisms governing protein-protein interactions from synthetic binding interfaces. Curr. Opin. Struct. Biol. 18, 499–506 (2008).

    Article  CAS  Google Scholar 

  44. Jones, S. & Thornton, J.M. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20 (1996).

    Article  CAS  Google Scholar 

  45. Lunde, B.M., Moore, C. & Varani, G. RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell Biol. 8, 479–490 (2007).

    Article  CAS  Google Scholar 

  46. Messias, A.C. & Sattler, M. Structural basis of single-stranded RNA recognition. Acc. Chem. Res. 37, 279–287 (2004).

    Article  CAS  Google Scholar 

  47. Bergman, N.H., Lau, N.C., Lehnert, V., Westhof, E. & Bartel, D.P. The three-dimensional architecture of the class I ligase ribozyme. RNA 10, 176–184 (2004).

    Article  CAS  Google Scholar 

  48. Saville, B.S. & Collins, R.A. A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria. Cell 61, 685–696 (1990).

    Article  CAS  Google Scholar 

  49. Chao, J.A., Patskovsky, Y., Almo, S.C. & Singer, R.H. Structural basis for the coevolution of a viral RNA-protein complex. Nat. Struct. Mol. Biol. 15, 103–105 (2008).

    Article  CAS  Google Scholar 

  50. Hogg, J.R. & Collins, K. RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation. RNA 13, 868–880 (2007).

    Article  CAS  Google Scholar 

  51. Keryer-Bibens, C., Barreau, C. & Osborne, H.B. Tethering of proteins to RNAs by bacteriophage proteins. Biol. Cell 100, 125–138 (2008).

    Article  CAS  Google Scholar 

  52. Piekna-Przybylska, D., Liu, B. & Fournier, M.J. The U1 snRNA hairpin II as a RNA affinity tag for selecting snoRNP complexes. Methods Enzymol. 425, 317–353 (2007).

    Article  CAS  Google Scholar 

  53. Kabat, E.A. & Wu, T.T. Attempts to locate complementarity-determining residues in the variable positions of light and heavy chains. Ann. NY Acad. Sci. 190, 382–393 (1971).

    Article  CAS  Google Scholar 

  54. Fellouse, F.A. et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J. Mol. Biol. 373, 924 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank members of the Piccirilli, Koide and Kossiakoff laboratories for assistance with phage display and for insightful comments; D. Lilley and T. Wilson for helpful discussions regarding the VS ribozyme; and V. Torbeev, I. Dementieva, P. Rice and V. Tereshko for assistance with crystallography. This work was funded by Howard Hughes Medical Institute (J.A.P.), US National Institute of General Medical Sciences Medical Scientist National Research Service Award no. 5 T32 GM07281 (Y.K.) and US National Institutes of Health grants R01-GM72688 and U54-GM74946 (to S.K. and A.A.K.) and R01-GM61835 (to D.P.B.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (grant 085P1000817).

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  1. David M Shechner

    Present address: Current address: Broad Institute and Harvard University Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Chemistry, University of Chicago, Chicago, Illinois, USA

    Yelena Koldobskaya & Joseph A Piccirilli

  2. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    Erica M Duguid, Nikolai B Suslov, Shohei Koide, Anthony A Kossiakoff & Joseph A Piccirilli

  3. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    David M Shechner & David P Bartel

  4. Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    David M Shechner & David P Bartel

  5. Department of Chemistry, University of Central Florida, Orlando, Florida, USA

    Jingdong Ye

  6. Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada

    Sachdev S Sidhu

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  1. Yelena Koldobskaya
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Contributions

All authors designed research; Y.K., E.M.D., D.M.S. and N.B.S. performed experiments; Y.K., E.M.D., D.M.S., S.K., A.A.K. and J.A.P. analyzed data; Y.K., E.M.D., D.M.S., N.B.S., D.P.B., S.K., A.A.K. and J.A.P wrote the paper.

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Correspondence to Anthony A Kossiakoff or Joseph A Piccirilli.

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Koldobskaya, Y., Duguid, E., Shechner, D. et al. A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol 18, 100–106 (2011). https://doi.org/10.1038/nsmb.1945

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