Abstract 2819 Poliovirus 3CD Protein Fluctuates between Compact and Extended Conformations in Solution

The poliovirus nonstructural protein 3CD is composed of two domains named 3C and 3D, separated by a 7-8-residue linker.The N-terminal 3C domain possesses protease activity and the 3D protein alone is an RNA-dependent RNA polymerase (RdRp).Intriguingly, the 3CD precursor does not have RdRp function, but 3CD works as a better protease than 3C.Although the X-ray crystal structure of 3CD has been obtained, it cannot explicitly explain the higher protease activity of 3CD compared to 3C.According to previous molecular dynamics (MD) studies and verified by small-angle X-ray scattering experiments (SAXS), there are transient domaindomain interactions that may allow 3CD to adopt extended and compact conformations.To probe these potential 3C-3D interactions, we generated amino acid substitutions at key locations (i.e.D32A, K60A, K78A, S177A, and K531A) and characterized these protein variants using SAXS, Analytical Ultra Centrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) Spectroscopy and other biophysical techniques on 3CD proteins.Preliminary SAXS results, including changes to the radii of gyration (Rg) and solvent envelopes derived from DENSS, were consistent with variants favoring extended conformations.AUC also indicated changes to the frictional ratio, which would also be suggestive with a change to the conformational equilibrium.Altogether, these results provide compelling evidence that there are different conformations of the 3CD protein in solution.The 3CD conformations and 3C-3D domain interactions may contribute to the functional differences between 3CD and 3C/3D, which these variants may help to verify.