Talk abstracts

Talk on Saturday 11:00-11:20am submitted by Jingdong Ye

Selection and Cocrystallization of FAB-P4P6 Complex: towards Chaperone-Assisted RNA Crystallography

Jingdong Ye (HHMI, Department of Biochemistry and Molecular Biology), Valentina Tereshko (Department of Biochemistry and Molecular Biology), John K. Frederiksen (Department of Biochemistry and Molecular Biology), Joseph A. Piccirilli (HHMI, Department of Biochemistry and Molecular Biology)

Abstract:
Comparing to protein, RNA crystallization is a much more challenging task. Among the extra features presented in RNA, the most prominent is the negatively charged backbone that adopt intricate scaffold and might make crystal packing more difficult. We are trying to tackle this problem by co-crystallize the RNA with its Antigen Binding Fragment (FAB). Using a reduced codon antibody FAB library displayed on the M13 phage, we have selected FABs that bind to Delta C209 P4-P6 domain of Tetrahymena Group I intron. Two FABs, FAB2 and FAB5, bind to the Delta C209 with affinities of 50 nM and 30 nM, respectively. These FABs are highly specific and do not bind to BP, a Delta C209 mutant, in which tertiary RNA folding has been disrupted. Furthermore, the binding between these FABs and Delta C209 is diminished when [Mg2+] was reduced to zero, showing that these FABs bind to the tertiary structure of Delta C209. FAB2 was cocrystallized with Delta C209 and the structure was solved at 1.95 Å resolution. The Fe-EDTA footprinting assay and the crystal structure reveal that FAB2 does not alter the overall folding of Delta C209 either in solution or in crystal. The crystal structure also shows that, with direct and water-mediated hydrogen bonding network, FAB2 helps Delta C209 achieve its native folding with fewer innersphere coordinated magnesium ions. The protein participated crystal contacts account for 61% of the buried surface area and we expect this method will facilitate the crystallization of RNA by providing crystal contacts and lending structure stability.

Keywords: Chaperone-Assisted RNA Crystallization, RNA Antibody, Phage Display