Monitoring the Conformation of the Sba1/Hsp90 Complex in the Presence of Nucleotides with Mn(II)-Based Double Electron–Electron Resonance

Angeliki Giannoulis,Tamar Unger,Shiran Amir,Daniella Goldfarb,Akiva Feintuch

doi:10.1021/acs.jpclett.1c03641

Abstract

Hsp90 is an important molecular chaperone that facilitates the maturation of client proteins. It is a homodimer, and its function depends on a conformational cycle controlled by ATP hydrolysis and co-chaperones binding. We explored the binding of co-chaperone Sba1 to yeast Hsp90 (yHsp90) and the associated conformational change of yHsp90 in the pre- and post-ATP hydrolysis states by double electron–electron resonance (DEER) distance measurements. We substituted the Mg(II) cofactor at the ATPase site with paramagnetic Mn(II) and established the binding of Sba1 by measuring the distance between Mn(II) and a nitroxide (NO) spin-label on Sba1. Then, Mn(II)–NO DEER measurements on yHsp90 labeled with NO at the N-terminal domain detected the shift toward the closed conformation for both hydrolysis states. Finally, Mn(II)–Mn(II) DEER showed that Sba1 induced a closed conformation different from those with just bound Mn(II)·nucleotides. Our results provide structural experimental evidence for the binding of Sba1 tuning the closed conformation of yHsp90.

Highlights

Hsp[90] is an important molecular chaperone that facilitates the maturation of client proteins
Heat shock protein of 90 kDa (Hsp90)’s function is intimately coupled to ATP binding and hydrolysis[5,6] and an associated cycle of conformational changes.[7−9] more than 20 co-chaperones have been found to finely regulate eukaryotic Hsp90.10−12 Hsp[90] in all organisms is a flexible homodimer with each monomer consisting of three highly conserved domains: the amino-terminal domain (NTD) where the ATPase site is found,[13] the middle domain (MD) that is important for ATP hydrolysis and binding of clients, and the carboxyl-terminal domain (CTD) that is responsible for dimerization of the two monomers[14,15] and contains a binding site for some co-chaperones[12] (Figure 1A)
The structural evidence for this plasticity has been obtained from fluorescence resonance energy transfer (FRET),[18−23] single-angle X-ray scattering (SAXS),[24] X-ray crystallography/cryo-electron microscopy,[25−28] nuclear magnetic resonance (NMR),[29,30] and recently double electron−electron resonance (DEER)[31] experiments