Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Nucleotide-dependent conformational changes in the AAA ATPase p97 and a disease-causing mutant revealed by Cryo-Electron Microscopy and Mass Spectrometry     (#324)

Ashish Sethi 1 , Sepideh Valimehr 1 , Mohsen Kazemi 1 , Mohammad Hossein Tanipour 1 , Caryn Hepburn 2 , Ching-Seng Ang 1 , Nicholas Williamson 1 , Isabelle Rouiller 1
  1. University of Melbourne, Melbourne, VICTORIA, Australia
  2. Waters Corporation, NSW, Australia

p97/VCP (Valosin containing protein), an essential protein that is highly abundant and conserved throughout eukaryotes, interacts with a diverse group of adaptor proteins to perform a variety of essential cellular processes ranging from protein unfolding to DNA damage repair and membrane fusion. It belongs to the type II AAA (ATPases associated with diverse cellular activities) family and plays an essential role in cellular homeostasis by regulating mitochondrial-associated degradation (MAD), endoplasmic reticulum-associated degradation (ERAD), chromatin-associated degradation, endosomal trafficking and autophagy. Mutations found at the interface between two main domains; the N- and the D1 domain of p97 have been associated in a number of neurodegenerative diseases1 and cancer. How these pathogenic mutations change the cellular function of p97 without altering its structure or the functional hexameric biological unit assembly remains unknown. Using a combination of single-particle Cryo-Electron Microscopy (Cryo-EM) and Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), we reveal a better understanding of the conformational changes that p97 undergoes during the ATPase cycle2,3, and importantly, how disease-causing mutations deregulate the movement of the N-terminal domain in p97 upon nucleotide binding. Our results provide a molecular basis for understanding the alterations in the interactions between the N-, D1 and D2- domains in p97 wild type (WT) and a variant p97 R155P (a disease-causing mutation localized at the N- and D1-domain interface) upon nucleotide binding. Here, we explain why disease-causing mutations in p97 have altered the ADP-bound form of the enzyme to conformationally look more like an ATP-bound state, which correlates with the disease severity. Our work provides mechanistic insight into the regulation of movement of the N-domain and describes the interdomain communication associated with nucleotide binding, which provides a potential implication in modulating the enzyme activity for better therapeutics against neurodegenerative diseases and cancer.

  1. Halawani, D., LeBlanc, A.C., Rouiller, I., Michnick, S.W., Servant, M.J., Latterich, M. (2009) Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation. Molecular and cellular biology 29, 4484-4494.
  2. Rouiller, I., Butel, V.M., Latterich, M., Milligan, R.A., Wilson-Kubalek, E.M. (2000) A major conformational change in p97 AAA ATPase upon ATP binding. Molecular cell 6, 1485-1490.
  3. Rouiller, I., DeLaBarre, B., May, A.P., Weis, W.I., Brunger, A.T., Milligan, R.A., Wilson-Kubalek, E.M. (2002) Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nature structural biology 9, 950-957.