Structural Studies using Cryo-EM to Unravel Mechanistic Details of p47 Binding To p97

Abstract

The AAA+ ATPase p97 ( Cdc48/VCP) is associated with the regulation of multiple cellular activities leading to protein homeostasis. p97 derives energy from ATP hydrolysis to bring about a conformational change of the p97 hexamer which enables downstream substrate processing. The substrate recognition and ubiquitin-dependent proteostasis pathways are under the control of various co-factors or adaptor proteins which form crucial p97-subcomplexes. This Mg+2 dependent ATPase has an N-terminal domain, followed by a conserved D1 and catalytic D2 ATPase domain and an unstructured C-terminal tail. The adaptor proteins of the p97 interact with either the N-domain or the C-terminal tail, resulting in a modulation of the ATPase activity of p97, thereby affecting its cellular function. Heterozygous missense mutations of human p97 have been detected in numerous neurodegenerative diseases, such as IBMPFD and familial amyotrophic lateral sclerosis. The disease mutations of the p97 are mostly clustered on the N-domain or the connection between N- and D1-domain. The mutation of R155H on the N- domain is one of the highest mutated sites p47, an essential cofactor to the p97-mediated membrane reassembly of the Golgi apparatus and nuclear envelope at the end stage of mitosis, has a preference to bind the R155H mutant over the wild-type p97. The p47 cofactor binding also alters the ATPase activities of both the p97 and p97 R155H. The mechanistic detail of the disease mutation of p97 R155H on the p47 binding has not fully investigated yet. To deeply understand the molecular details, we assembled the proteins in vitro and determined the structures to answer disease-relevance of the complex.