The homohexameric AAA ATPase p97 plays an important role in protein homeostasis in the cell and has consequently been identified as a target for cancer treatment. Single amino acid mutations in this protein cause Inclusion Body Myopathy with early-onset Paget disease and Frontotemporal Dementia (IBMPFD) and Amyotrophic Lateral Sclerosis (ALS) and it can affect the muscles, bone and brain. One of the most clinically relevant mutation is R155P in which Arginine 155 is replaced by Proline. This variant forms hexamer the same as the wild type and has an increased ATPase activity. However, the effect of this mutant on protein structure is not known.
Using single particle cryo-EM, we aimed to define the structural impact of this mutation on p97 in order to explain the increased ATPase activity. In the first instance, we screened different sample preparation conditions in order to get p97 in ice and in multiple orientation, including side views. Results of this screen will be presented. Best conditions were obtained with 3mgl/m protein and 0.02% detergent (NP40). Cryo-EM data was collected using Talos Arctica, equipped with K2 Summit camera (Gatan, Inc) at the Bio21 institute. We obtained a preliminary map at 3.8Å of p97R155P after incubation with ATPγS. Interestingly this map shows p97 in a novel conformation.
The novel conformation presented allows further understanding of p97 catalytic cycle, that is what are the conformation adopted by p97 during the ATPase cycle are associated with the motion of the different domains of the protein. The knowledge of the mechanism regulating p97 is important for drug design for treatment both of cancer and neurodegenerative diseases.