Book of Abstracts: Albany 2007
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
1 Howard Hughes Medical Institute; Department of Chemistry, University of Chicago, 920 E. 57th St., Chicago, IL 60637