Pathogenic mutation impairs functional dynamics of Hsp60 in mono- and oligomeric states.

Mitochondrial chaperonin Heat Shock Protein 60 kDa (Hsp60) oversees the correct folding of client proteins in cooperation with Hsp10. Hsp60 monomers M first form 7-meric Single rings (S), which then pair into 14-meric Double rings (D) that accommodate clients in their lumen. Recruitment of 7 Hsp10 molecules per pole yields a sealed 28-meric Football-shaped complex (F). ATP hydrolysis in each Hsp60 unit drives client folding and F disassembly. The V72I mutation in hereditary spastic paraplegia form SPG13 impairs Hsp60 function despite being distant from the active site. We here investigate this impairment with atomistic molecular dynamics (MD) simulations of M, S, D, and F for both WT and mutant Hsp60, considering catalytic aspartates in D and F in different protonation states (even simulating one such state of D post-hydrolysis). Our findings show that-as observed experimentally-V72I rigidifies Hsp60 assemblies, significantly impacting internal dynamics. In monomers, V72I introduces a new allosteric route that bypasses the ATP binding site and affects mechanisms driving reactivity. These insights highlight a multiscale complexity of Hsp60 that could inspire the design of experiments to better understand both its WT and V72I variants.