Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.

Abstract

p97/VCP is a homohexameric AAA-ATPase associated with diverse cellular activities such as DNA repair and endoplamic reticulum-associated degradation (ERAD). The diversity of its functions lies within the interaction with different adaptor proteins such as p47 and Ufd1/Npl4 (UN) heterodimer. By binding to the major adaptor protein binding site at the N-terminal domain of p97/VCP, adaptor proteins direct p97/VCP to different sites and different activities in the cell, utilizing the energy generated by ATP hydrolysis in the D2 domains of p97/VCP. This process should be regulated. Competition binding assays by Chia et al., 2012, showed that ATP binding to D1 was able to increase p97/VCP affinity to UN compared to p47. In this research, we aimed to gain further insight on Chia et al. findings by mutating critical ATP binding residues, K251A, R359A and H384A in D1 and D478A, K524A and K658A in D2, and comparing the binding affinities of UN to wild type and mutant p97/VCP by SPR. Due to time limitations, we could focus only on one of the mutants, K251A. However, the mutant protein did not generate hexamers for us to proceed with binding assays. Future hexamerization and binding studies will be done with the mutants.