Understanding Protein Folding Mechanisms of Chaperone Proteins Using Cryo-EM

Abstract

Chaperone proteins are central to protein quality control and cell signaling. Three major classes of chaperones include foldases, holdases and disaggregases. Foldases aid proteins in folding to their native state, as well as prevent them from misfolding, a process that is regulated by ATP hydrolysis. Holdases are ATP-independent chaperones that respond to cellular stress, such as heat shock and oxidation, and protect proteins from aggregation. Disaggregases rescue aggregated proteins, resolubilize them and hand them off to foldases to be refolded. Together, these three classes of chaperones regulate many cellular processes and maintain proteostasis. In this study, three chaperones were characterized using electron microscopy: Hsp104 (disaggregase), Get3 (holdase), and FKBP51 (foldase). Hsp104 is a double ring AAA+ protein that is active as a hexamer and is capable of pulling apart amorphous aggregates and amyloid fibrils. AAA+ proteins have conserved ATP hydrolysis pockets and conserved translocation mechanism to thread polymers through the central channel. Previous structural work of Hsp104 and other AAA+ proteins has been limited to low resolution due to the dynamic nature of the hexamer. In this study, we have determined three high-resolution structures of Hsp104. First, an open, asymmetric spiral conformation of Hsp104:AMPPNP to 5.6Å, which revealed a large hexamer seam with a unique hetero-AAA+ interaction between the Nucleotide Binding Domain (NBD) 1 and 2. Next we determined two structures of Hsp104 bound to substrate in the presence of ATPS; the closed (4.0Å) and extended (4.1Å) states. The critical substrate binding tyrosine loops are arranged in a two-turn spiral around the substrate, showing direct interactions between the tyrosine and polypeptide chain. The closed state has only five of six protomers engaged, whereas in the extended state all six protomers are engaged with a translocation step of 6-7Å between states. From our structures, we propose a processive rotary translocation mechanism between the closed and extended states, with a non-processive step from the open to closed states upon substrate binding. These structures give insight into the translocation of the highly conserved AAA+ class of proteins. In another study, the holdase activity of Get3 was investigated. Get3 is important for ATP-dependent Tail Anchored-binding protein membrane insertion in yeast. We established that upon oxidative stress, Get3 undergoes a structural rearrangement from a reduced, ATP-dependent dimer, to an oxidized, ATP-independent tetramer with a general chaperone function. Using negative stain-EM we further characterized the Get3ox tetramer to determine that it has a ‘W’ shape, revealing a hydrophobic patch to bind substrates. Lastly, we studied the interaction between Hsp90 and its cochaperone, FKBP51, an immunophilin that is upregulated in cancer and Alzheimers disease. We determined that Hsp90 binds FKBP51 in its closed, ATP-bound state, a complex that is stabilized by another cochaperone, p23. Negative stain EM establishes the contacts between Hsp90 and FKBP51, which could be used as potential drug targets with improved specificity from Hsp90 inhibitors. We have determined the structures of three different chaperone classes, showing the very dynamic nature of molecular chaperones and elucidating functional mechanisms of these chaperones. Overall this thesis work has made advancements in all three areas studied, utilizing structure determination to correlate with function.