Crystalizing Structured RNAs Using a Surface‐Entropy‐Reduced Fab as a Crystallization Chaperone

Abstract

Our lab studies the molecular mechanisms of regulatory RNAs elements by determining their atomic resolution structure using x‐ray crystallography. However, RNA crystallization presents many challenges. One strategy to meet these challenges is the use of a protein chaperone to assist RNA crystallization. Antibody fragments (Fabs) provide one type of crystallization chaperone, and we have used these successfully for RNA crystallization in two ways. In one approach, a Fab‐binding‐RNA epitope is grafted into the RNA of interest to create a binding site for the cognate Fab crystallization chaperone. To improve this method, I performed surface entropy reduction on the Fab framework to make a version of our crystallization chaperone that is more suited to well‐ordered crystal packing. Using structural analysis software as a guide, I made twelve surface‐entropy‐reduced (SER) mutant versions of the Fab framework, in which patches of bulky, flexible residues are mutated to alanine. To test the performance of these mutants, I carried out crystallization trials of their complexes with the yjdF RNA, a novel bacterial riboswitch that binds to aza‐aromatic compounds. The wild type Fab (wt BL3‐6) and many of the mutants were able to crystalize with the riboswitch and gave diffraction datasets with 3.5 – 3 angstrom resolution range, and the structure is currently being solved. Using the mutants, I have also crystalized a second RNA target, PEMV2, which is a viral cap‐independent‐translation‐element. This RNA crystalized with wild type but could only diffract to 2.75 angstrom resolution with the aid of a mutant. I am in the process of solving this structure which will give valuable hints to the mechanism of action of this class of viral cap‐independent‐translation‐elements. As I refine this crystallization chaperone technology, I will also apply it to a notoriously difficult crystallization target, domain V of the human polio virus IRES.Support or Funding InformationNIH, HHMI, The University of Chicago