Molecular Insights into the A. thaliana CDC48-NPL4-UFD1 Complex

  • Brandon D. Zahodnik-Huntington

Student thesis: Master's Thesis

Abstract

The maintenance of protein homeostasis as a response to changing external conditions is crucial for cellular survival and proper function. Since plants cannot adapt by changing location, their need for a rapid intracellular response is accentuated. The AAA ATPase CDC48 maintains protein homeostasis in conjunction with NPL4 and UFD1 by coupling ATP hydrolysis with mechanical force to extract and unfold ubiquitylated proteins from organelle membranes, chromatin, or protein complexes. Our bioinformatic analysis revealed considerable domain and binding motif differences in A. thaliana NPL4 compared to its orthologs in animals and fungi. Using ITC, MST, and SEC-MALS, we found that NPL4 and UFD1 did not heterodimerize, NPL4 bound to CDC48A in the absence of UFD1, and the complex was not stable in vitro. Additionally, we provided the first medium-high-resolution reconstructions of CDC48A in both an AMP-PNP bound and apo state, using cryo-EM. AMP-PNP bound CDC48A was reconstructed in both a tense (3.3 Å) and relaxed (3.5 Å) conformation with the N domain was positioned above or coplanar with the D1 ring, respectively. Our heterogeneity analysis using CryoDRGN revealed continuous flexibility of the N domains between the two conformations. The apo state was reconstructed as a single conformation at 4.4 Å resolution. A cryo-EM reconstruction of the complex was also obtained at a resolution of ~6 Å, which showed expected cofactor stoichiometry and binding positions. Through our efforts, we have observed differences in the interaction between A. thaliana CDC48A and its cofactors UFD1 and NPL4 that may correspond to functional differences between kingdoms.
Date of AwardJul 2021
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorStefan Arold (Supervisor)

Keywords

  • CDC48, Cofactors, cryo-EM, Protein homeostasis, UFDI, NPL4

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