Understanding the Mechanism of Disaggregation and Unfolding of the hsp100 Family Through cryo-EM Structure Determination
Rizo, Alexandrea
2022
Abstract
Proteins in the cell can become misfolded, leading to toxic aggregates. These toxic aggregates, which can increase during cellular stress and aging, are hallmarks of many neurodegenerative diseases, including Alzheimer’s and Parkinson’s Disease. The Heat-Shock Protein (Hsp) 100 family rescues these aggregates and returns proteins to their unfolded states by acting as unfoldases and disaggregases. Hsp100s are hexameric protein assemblies that translocate substrates through their central channel via cycles of ATP hydrolysis. Previous studies by x-ray crystallography revealed the domain architecture, including the conserved AAA+ (Atpases Associated with diverse Activities) structure. However, due to limitations of crystallography, the functional hexameric assemblies were not captured. Early work by cryo-electron microscopy (cryo-EM) identified hexamer states but was limited due to applied symmetry in structural refinements and the use of inactive, substrate-free complexes. For my dissertation I determined structures of active, substrate-bound Hsp100 family members by cryo-EM to understand how these AAA+ machines unfold substrates. These structures provide details into the ATP hydrolysis-dependent conformational changes that occur upon the addition of substrate and the interactions that occur with substrate in the central channel. To understand how these complexes function in various organisms, I solved structures of Hsp104 from yeast, ClpB and ClpAP from bacteria, and Skd3 from eukaryotes. The structures revealed that Hsp100s form a left-handed hexameric spiral with conserved tyrosine pore loops lining the central channel when bound to the substrate. By analyzing the nucleotide states of the different protomers, we propose a model for ATP hydrolysis. This triggers the nucleotide-binding domains to undergo conformational changes at the spiral seam interface driving substrate translocation via a ratchet-like rotary mechanism around the hexamer. These conformational changes are observed in numerous structures highlighting a conserved motor mechanism of the Hsp100 family that can be tuned specifically for each species. Due to the high resolution of the ClpB reconstruction, I determined specific interactions that the pore loops and the N-terminal domain make with substrate in the central channel aiding in both substrate recognition and delivery. These complexes also contain specie specific domains and elements, including the IGL-loops in ClpA and ankyrin repeat domain in Skd3, which help in the allosteric control of substrate unfolding that supports the overall function in proteostasis. These structures have comprehensively explained how the Hsp100 family functions as powerful unfoldases and disaggregases and uncovered a conserved mechanism within various organisms.Deep Blue DOI
Subjects
Hsp100 family cryoEM unfoldase disaggregase proteostasis
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.