Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Chaperone-Assisted RNA Crystallography: Selection and Co-crystallization of a Fab-ΔC209 P4-P6 complex

Despite great progress in the past decade, RNA crystallization remains challenging. Here we present a new strategy to overcome this problem: co-crystallization of RNA with an Antigen Binding Fragment (Fab) derived from in vitro selection via phage display. Using a reduced codon antibody Fab library displayed on the M13 phage, we have obtained two Fabs that bind with low-nanomolar affinities to the ΔC209 P4-P6 domain derived from the Tetrahymena group I intron. These Fabs are highly specific and bind much more weakly to a P4-P6 mutant that cannot form long-range tertiary interactions. Binding requires magnesium, suggesting that these Fabs bind the tertiary structure of ΔC209 P4-P6. Fab2 was co-crystallized with ΔC209 P4-P6 and the structure was solved at 1.95 Å resolution. Both the crystal structure and hydroxyl radical footprinting data indicate that Fab2 does not alter the overall folding of ΔC209 P4-P6. The Fab2-ΔC209 P4-P6 structure reveals a complicated RNA-binding strategy achieved by arrays of peptide loops presented on a shallow surface. By using direct and water-mediated hydrogen bonding networks, Fab2 helps ΔC209 P4-P6 achieve its native structure with fewer innersphere coordinated magnesium ions. The main crystal contacts are mediated by Fabs. We expect that chaperone-assisted RNA crystallography (CARC) will facilitate the crystallization of RNA by providing crystal contacts and initial phase information, and possibly by improving structure stability.

Jing-Dong Ye 1
Valentina Tereshko2
John K. Frederiksen2
Akiko Koide2
Frederic A. Fellouse3
Sachdev S. Sidhu3
Shohei Koide*2
Anthony A. Kossiakoff*2
Joseph A. Piccirilli*1, 2

1 Howard Hughes Medical Institute; Department of Chemistry, University of Chicago, 920 E. 57th St., Chicago, IL 60637
2 Department of Biochemistry and Molecular Biology, University of Chicago, 920 E. 57th St., Chicago, IL 60637
3 Department of Protein Engineering, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080, USA

Tel: 773-702-4718
Fax: 773-702-0271
Email: jpicciri@uchicago.edu