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

Abstract

AbstractHeat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. MonomersMof Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings (S), then pairs ofSinterface equatorially to form 14-meric Double rings (D) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turnsDinto a 28-meric Football-shaped complex (F). Sequential hydrolysis of ATP present in each Hsp60 unit ofFfinally drives client folding andFdisassembly. 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 ofM,S,D, andFfor both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates inDandFmodelled in three different protonation states. Additionally,Din one protonation state was modelled post-hydrolysis (total production time: 36 µs). 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 isolatedMunits right up toF, 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.