Structural Analysis of Arabidopsis thaliana CDC48A ATPase using Single Particle Cryo-Electron Microscopy

  • Lila A. Aldakheel

Student thesis: Master's Thesis

Abstract

Cdc48A and its human homologue P97 are from ATPase family, which play a variety of roles in cellular activates and it has a crucial involvement in protein quality control pathways. It is best known for its involvement in endoplasmic reticulum associated protein degradation (ERAD), where it mediates the degradation of the aggerated or misfolded proteins by the proteasome. Considering the multiple functions of Cdc48A in many protein regulatory processes, it is a potential therapeutic target for neurogenerative diseases and cancer. Cdc48A polypeptide comprises N domain, followed by D1 and D2 domains respectively that are joined by linkers, whereas functionally it forms a homo hexameric complex. Since Cdc48A is from the ATPase family, it uses the ATP hydrolysis to generate a mechanical force with its co-factors to perform its functions. There are many cofactors that interact with Cdc48A and two of them are Ufd1-NpI4 which in turn interact with ubiquitinated proteins from the ER membrane. The mechanism linking the conversion of the energy of ATP hydrolysis into mechanical force and unfolding the substrate is vague. My aim is to reconstruct a first 3D- model of plant Cdc48A using single particle cryo-EM, which serves the basis to conduct more detailed mechanistic studies towards substrate unfolding and threading/unfolding in the future. In general, results showed one defined structure of cdc48A at ~ 9.8 Å, which is the ADP-ATP conformation. Although another other structure was also resolved at ~ 8.9 Å, it was hard to characterize due to its dissimilarity with known structures of Cdc48A homologues and thus requires further characterization.
Date of AwardMay 2019
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorStefan Arold (Supervisor)

Keywords

  • cryo-EM
  • CDC48A
  • P97
  • single particle analysis

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