Biology subjects

Myasnikov, A.

Publications and source records attributed to Myasnikov, A..

3 recordsLinked to original sources

bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90s many "client" proteins3-7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8-13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

biochemistry↗

bioRxiv2020

Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates

TRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of "client" proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highlight a client binding mechanism conserved throughout Hsp90s that transcends the need for cochaperones and provide molecular insights into how TRAP1 modulates protein folding within mitochondria. Our structures also suggest a potential role for TRAP1 in Fe-S cluster biogenesis and mitochondrial protein import and will guide small molecule development for therapeutic intervention in specific TRAP1 client interactions.

biophysics↗

bioRxiv2019

High-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit and validation of nucleotide modifications

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides positions, a task that has previously been accomplished by X-ray crystallography. Here we present a cryo-electron microscopy (cryo-EM) structure of Escherichia coli (E. coli) 50S subunit at an average resolution of 2.2[A] as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for identification of post-transcriptional rRNA modifications using a cryo-EM map. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

biochemistry↗