UCSF

The maintenance of protein homeostasis (proteostasis) is essential in all living organisms and requires a robust network of pro-folding and pro-degradation factors. Among others, proteostasis machinery includes molecular chaperones, which promote the folding of newly-synthesized and transiently misfolded proteins, and the proteasome, which degrades misfolded or otherwise aberrant proteins. These machines frequently exhibit pronounced conformational variability and are often coupled to co-chaperones and adapter proteins, likely to accommodate the processing of diverse substrates and enable specific cellular functions. While decades of biochemistry and cell biology have established the importance of these systems, direct observation of these macromolecular complexes in functionally relevant states has only recently been enabled by seminal hardware and software advancements in high-resolution single-particle cryo-electron microscopy (cryo-EM). In addition to making routine the structure determination of almost any macromolecule of interest, this technique allows for the classification of particles based on compositional and conformational variability, and is thus uniquely suited to uncover the complicated structural dynamics characteristic of most proteostasis machinery. My doctoral work has focused on using cryo-EM to visualize challenging targets in the proteostasis network, with an emphasis on uncovering rare or dynamic states central to the understanding of these machines. The first chapter of this dissertation reviews how the structure and function of the human AAA+ segregase p97/VCP is regulated by a large and structurally and mechanistically diverse set of adapter proteins critical for its function. Mirroring the themes of my doctoral work, we establish that single particle cryo-EM has begun to reveal the molecular basis for many p97-adapter interactions, and suggest that this technique, coupled with advances in computational structure prediction and in situ structural biology, will continue to play an important role in advancing understanding of this essential and multifunctional protein complex. The second chapter reports high-resolution cryo-EM structures of p97 in complex with UBXD1, a particularly enigmatic adapter implicated in the autophagic clearance of damaged organelles and other functions. We show that UBXD1 binding potently inhibits p97 ATPase activity and structurally remodels p97 using an extensive and unprecedented set of interactions. These interactions split the stable p97 hexamer into an open ring conformation that potentially enables unique modes of substrate processing. The third chapter describes the reaction cycle of human mitochondrial heat shock protein 60 (mtHsp60), a conserved molecular chaperone that promotes the folding of proteins in the mitochondrial matrix. Using cryo-EM image processing techniques including symmetry expansion and focused classification, we uncover novel conformations of this dynamic machine that provide a structural rationale for the simultaneous substrate- and co-chaperone-binding activity observed in some mtHsp60 intermediates. These results likely apply to all mtHsp60 homologs. The fourth chapter describes how the human 20S proteasome is remodeled by a suboptimal peptide activator derived from the consensus hydrophobic, Tyr, any amino acid (HbYX) sequence. We show that this peptide binds in multiple conformations in the 20S alpha pockets, inducing a radial expansion of alpha subunit N-terminal regions that are on-pathway to complete activation. While some disordering of the 20S gate is observed, indicating partial activation, it appears that a consensus HbYX sequence is necessary to completely open the gate and allow substrates to access the interior proteolytic active sites.