How a pathogenic mutation impairs Hsp60 functional dynamics from monomeric to fully assembled states

Abstract

Heat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. Monomers M of Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings (S), then pairs of S interface equatorially to form 14-meric Double rings (D) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turns D into a 28-meric Football-shaped complex (F). Sequential hydrolysis of ATP present in each Hsp60 unit of F finally drives client folding and F disassembly. Equatorial domain mutation V72I occurs in SPG13, a form of hereditary spastic paraplegia: while distal to the active site, this severely impairs the chaperone cycle and stability. To understand the molecular bases of this impairment we have run atomistic molecular dynamics (MD) simulations of M, S, D, and F for both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates in D and F modelled in three different protonation states. Additionally, D in one protonation state was modelled post-hydrolysis (total production time: 36 {micro}s). By combining complementary experimental and computational approaches for the analysis of functional dynamics and allosteric mechanisms, we consistently find that mutation V72I significantly rewires allosteric routes present in WT Hsp60 across its complexes, from isolated M units right up to F, rigidifying them--as observed experimentally--by introducing a direct allosteric link between equatorial and apical Hsp60 domains that bypasses the ATP binding site (wherein we observe the alteration of mechanisms driving reactivity). Our results reveal a multiscale complexity of functional mechanisms for Hsp60 and its pathogenic mutant, and may lay the foundation for the design of experiments to fully understand both variants.