Characterization of Coronaviruses Mac1 Mutant Proteins

Abstract

The coronaviruses macrodomain, also called Mac1, in the nonstructural protein 3 (nsp3) has two important functions: it binds to a protein in the acidic residues (glutamate and aspartate), and hydrolyzes as a mono-ADP-ribose (MAR) from a protein including aspartic acid and asparagine. The biochemistry results of Mac1 protein showed their functions with PARPs. However, there are limited studies on the role of specific residues in the CoV macrodomain. Based on the crystal structure of Mac1 with binding affinity to ADP-ribose, here we focused on two mostly conserved amino acid residues of Mac1 that bind with ADP-ribose in all CoVs, N40, and D22, as mutants to alanine. We also attempted to produce an MHV-Mac1 purified protein, which was unsuccessful in the E. coli or the pET system. This thesis establishes a large-scale protein expression and purification in E. coli for both WT and point mutant CoV Mac1 proteins, which had previously been extremely difficult to purify. The CoVs-Mac1 proteins were characterized by a broad set of biochemical methods. We studied the structure of several Mac1 proteins by circular dichroism (CD). The differential scanning fluorimetry (DSF) and alpha screen assays were used to measure ADP-ribose binding. Finally, we also analyzed the ability of these proteins to remove ADP-ribose from a protein substrate.