Chemical Footprinting of Polymeric Structure of hnRNPA2 Low Complexity Domain

Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity domains. These low complexity sequences have been shown to induce phase transition in low salt buffer. When incubated at high concentration, certain of these low complexity domains polymerize into labile, amyloid-like fibers. I developed a chemical footprinting method to probe solvent accessible residues in the low complexity domain polymers. By acetylating protein side chains with N-acetylimidazole, and comparing the acetylation in native and denatured conformation by use of SILAC mass spectrometry, I generated an NAI footprint for hnRNPA2 polymers. I deployed this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, and offered evidence that its low complexity domain exists in a similar conformation as that described for recombinant polymers of the protein. To study the structure of the low complexity sequence in liquid-like droplets, I systematically mutated individual tyrosine or phenylalanine residues to serine, assayed the ratio of these mutants that partitioned into the droplet phase, and compared the results with their abilities to grow polymeric fibers from wild-type seeds. The same region which contained mutations impeding fiber growth were found to display decreased partitioning into liquid-like droplets. Additionally, the NAI footprint of hnRNPA2 in these liquid-like droplets appeared to be similar to the footprint found in fibers. These observations suggest that the hnRNPA2 low complexity domain adopts a similar structure in amyloid-like fibers and liquid-like droplets. Combining these results, my studies favor the perspective that cross-beta polymerization commonly drives the formation of hydrogels, the retention of low complexity domains trapped by hydrogels, the formation of liquid-like droplets, the partitioning of low complexity domains into existing liquid-like droplets, and the formation and maturation of RNA granules. In other words, my results provide evidence that the involvement of low complexity domains in the formation of RNA granules, liquid-like droplets and hydrogels all rely on one in the same phenomenon - cross-beta polymerization.