Crystallization of Mouse RIG-I ATPase Domain: In Situ Proteolysis

Abstract

RIG-I is a key pattern recognition receptor that recognizes cytoplasmic viral RNA. Upon ligand binding, it undergoes a conformational change that induces an active signaling conformation. However, the details of this conformational change remain elusive until high-resolution crystal structures of different functional conformations are available. X-ray crystallography is a powerful tool to study structure-function relationships, but crystallization is often the limiting step of the method. Here, we describe the in situ in-drop proteolysis of RIG-I that yielded crystals of the ATPase domain of mouse RIG-I suitable for structure determination.

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish

“…Both studies were primarily concerned with increasing the number of crystallizable protein fragments as part of a structural genomics effort. Several other preliminary protein crystallization notes and publications involving structure determination of proteins or domains thereof reported the use of in situ proteolysis (Gaur et al, 2004;Johnson et al, 2006;Taneja et al, 2006;Forsgren et al, 2009;Little et al, 2012;Civril and Hopfner, 2014;Kobayashi et al, 2014). At least in two cases, the importance of proteolysis for growing diffraction-quality crystals was a serendipitous discovery in that the solution was infected with a fungus that secreted the protease (Mandel et al, 2006;Bai et al, 2007).…”