Hereditary spastic paraplegia SPG13 mutation increases structural stability and ATPase activity of human mitochondrial chaperonin

Abstract

Human mitochondrial chaperonin mHsp60 is broadly associated with various human health conditions and the V72I mutation in mHsp60 causes a form of hereditary spastic paraplegia, a neurodegenerative disease. The main function of mHsp60 is to assist folding of mitochondrial proteins in an ATP-dependent manner. In this study, we unexpectedly found that mutant mHsp60V72I was more stable structurally and more active in the ATPase activity than the wildtype. Analysis of our recently solved cryo-EM structure of mHsp60 revealed allosteric roles of V72I in structural stability and ATPase activity, which were supported by studies including those using the V72A mutation. Despite with the increases in structural stability and ATPase activity, mHsp60V72I was less efficient in folding malate dehydrogenase, a putative mHsp60 substrate protein in mitochondria and also commonly used in chaperonin studies. In addition, although mHsp60V72I along with its cochaperonin mHsp10 was able to substitute the E. coli chaperonin system in supporting cell growth under normal temperature of 37 °C, it was unable under heat shock temperature of 42 °C. Our results support the importance of structural dynamics and an optimal ATP turnover that mHsp60 has evolved for its function and physiology. We propose that unproductive energy utilization, or hyperactive ATPase activity and compromised folding function, not mutually exclusive, are responsible for the V72I pathology in neurodegenerative disease.

Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly

“…1a). Importantly, the V72I mutation retains some client refolding activity in vitro 43 , suggesting that general features of the mtHsp60 chaperone cycle are preserved.…”

“…The hereditary spastic paraplegia variant mtHsp60 V72I forms stable heptamers and retains significant chaperone activity Wild type mtHsp60 heptamers are unstable in the absence of mtHsp10 in vitro, and readily dissociate into monomers at low concentration or temperature, or when incubated with nucleotide 39 , thereby complicating efforts to characterize the mtHsp60 chaperone cycle. To facilitate structural studies of mtHsp60, we focused on the previously identified mtHsp60 V72I variant that is associated with hereditary spastic paraplegia SPG13 26,29 , and is reported to have increased oligomeric stability 43 . Residue V72 (numbering corresponds to the mature mtHsp60 protein after cleavage of the mitochondrial import sequence) is located in the equatorial domain of mtHsp60 and packs into its hydrophobic core, but does not contact the ATP binding pocket (Fig.…”

“…We sought to determine the structural basis for progression through the nucleotide-and mtHsp10-dependent mtHsp60 chaperone cycle using a disease-associated mutant that increases oligomeric stability 26,28,43 . Cryo-EM structures of three mtHsp60 states in this cycle were determined: apo-mtHsp60, ATP-bound mtHsp60, and ATP-bound mtHsp60/mtHsp10.…”