Mechanism of the alteration in domain-domain communications in human p97/VCP atpase

Abstract

Human p97/VCP, an AAA+ ATPase, regulates various cellular activities by interacting with cofactor proteins, including ubiquitin-dependent protein quality control, Golgi-biogenesis, and endoplasmic reticulum-associated degradation (ERAD). Single amino-acid mutation of R155H on the N-domain is the most prevalent, leading to a rare degenerative disease MSP1. The p97 R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. We pursued biochemical characterization in combination with single-particle cryo-electron microscopy (cryo-EM) to study the interaction of p97^(R155H) mutant with its cofactor p47 and determined the structures of the p97^(R155H)-p47 complex in full length for the first time. In contrast to the wild type, p97^(R155H) occupies approximately 40% in the dodecameric form without nucleotide binding. The p97^(R155H) dodecamer does not bind to p47 or nucleotides and bears close resemblance to the inhibitor bound CB-5083:p97 structure, implying that this may be an inactive form. In the full-length p97^(R155H)-p47 complex structure, the p47 interacts through its UBX domain in an asymmetric manner to bind the p97^(R155H) N-domain with a preference for the N-up at the highest position. The structures also established that the arginine fingers might contribute to the elevated p97^(R155H) ATPase activity. Because the N-terminal domain is spatially far away from the nucleotide-binding site, the intermediate linkers may play a key role in these functional modulations. We went further to study functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The results showed the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity, regulating the domain-domain communication in p97 ATPase.

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