PhD Advisor: Professor Stefan T. Arold

Abstract:
The AAA+ ATPase CDC48 (p97/VCP in animals) is an essential unfoldase that extracts ubiquitinated proteins from membranes, complexes, and chromatin for degradation, acting through a diverse repertoire of substrate-targeting cofactors. Although the structure and mechanism of CDC48 have been characterised extensively in animals and yeast, the plant system had remained structurally uncharacterised despite CDC48's essential roles in plant-specific processes. This dissertation determined the structural and mechanistic basis of substrate processing by Arabidopsis thaliana CDC48A and identified the plant-specific adaptations that distinguish it from its animal and fungal counterparts. Reliable structural analysis of the CDC48A hexamer by cryo-electron microscopy (cryo-EM) first required overcoming the dissociation and preferential orientation common during specimen preparation. Investigating the principles underlying these effects established that the deliberate selection of thicker ice, combined with careful sample optimisation, preserved both the integrity and the angular distribution of the hexamer. Applying this approach, the structural and biochemical characterisation of CDC48A and its NPL4 and UFD1 cofactors revealed that, unlike the obligate heterodimer conserved in animals and yeast, Arabidopsis NPL4 and UFD1 bind CDC48A independently, with NPL4 alone sufficient to drive substrate unfolding. An evolutionary analysis suggested that this independent, modular mode of cofactor engagement may represent the ancestral state of the eukaryotic CDC48 system. To resolve the mechanistic consequences of this cofactor independence, the minimal complex of CDC48A–NPL4 was captured across multiple states of its substrate-processing cycle. NPL4 was found to engage the initiator ubiquitin within its conserved MPN groove in the absence of UFD1, with a plant-specific loop reconfiguring the arrangement of distal ubiquitins, while captured reaction intermediates provided insight into the mechanism of ATP hydrolysis across the D1 and D2 rings. Together, this work demonstrated that plant CDC48 is not a simplified version of its animal and fungal counterparts, but operates through a mechanistically distinct and structurally more minimal pathway for substrate recognition and processing. It establishes Arabidopsis CDC48A as a valuable system for understanding the minimal architecture underlying ubiquitin-dependent substrate unfolding.

Bio:
Brandon Zahodnik-Huntington is a PhD candidate in the Structural Biology and Engineering (StruBE) group at King Abdullah University of Science and Technology (KAUST), under the supervision of Professor Stefan T. Arold. His research uses high-resolution cryogenic electron microscopy (cryo-EM) alongside complementary biophysical and functional analyses to investigate the ubiquitin-proteasome system in plants, focusing on how the CDC48 unfoldase recognises and unfolds its targets. He received his BSc in Biology from the University of British Columbia (UBC), Canada, and his MSc in Bioscience from KAUST