A simple method for mapping the location of cross-β forming regions within protein domains of low sequence complexity

Abstract

Protein domains of low sequence complexity are unable to fold into stable, three-dimensional structures. In test tube studies, these unusual polypeptide regions can self-associate in a manner causing phase separation from aqueous solution. This form of protein:protein interaction has been implicated in numerous examples of dynamic morphological organization within eukaryotic cells. In several cases, the basis for low complexity domain (LCD) self-association and phase separation has been traced to the formation of labile cross-{beta} structures. The primary energetic force favoring formation of these transient and reversible structures is enabled by polypeptide backbone interactions. Short, contiguous networks of peptide backbone amino groups and carbonyl oxygens are zippered together intermolecularly by hydrogen bonding as described by Linus Pauling seven decades ago. Here we describe a simple, molecular biological method useful for the identification of localized, self-associating regions within larger protein domains of low sequence complexity. SignificanceThis study describes a molecular biological method for analyzing protein domains of low sequence complexity in search of segments that mediate self-association and consequent phase separation both in vitro and in vivo. Small regions allowing for self-association correspond to sequences that specify the formation of labile cross-{beta} structural order. When juxtaposed to the C-terminus of GFP, cross-{beta} prone regions suppress fluorescence. A tiled scan of overlapping fragments of the low complexity domain (LCD) of the TDP-43 RNA binding protein pinpointed an evolutionarily conserved sequence of twenty amino acids essential for self-association, phase separation and the formation of nuclear speckles. The screening method described herein should be useful for the analysis of any LCD believed to function via homotypic self-association.