Abstract
The human chaperonin complex is a ~ 1MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit.
Highlights
Recent advances in human genomics are revealing numerous pathogenic germline and somatic mutations [1], affecting chaperone genes [2]
We introduced the pathogenic mutation into the ortholog chaperonin from a hyperthermophilic archaeon, Pyrococcus furiosus (Pf), which shares 44% sequence identity with the human CCT5 [17]
The asymmetry observed in the differential scanning calorimetry (DSC) thermograms is evident for all proteins, and all peaks are skewed at temperatures below the transition midpoint (Tm), where the transition is less sharp and deviates more from the two-state fit, as expected for a transition coupled to dissociation [21,25]
Summary
Recent advances in human genomics are revealing numerous pathogenic germline and somatic mutations [1], affecting chaperone genes [2]. Elucidating mechanisms of functional deficits remains the bottleneck for pathogenetic analysis, which interferes with progress in understanding the mutations’ impact on protein homeostasis and therapeutics. Protein homeostasis is carried out mostly by the chaperoning system that, when faulty, leads to human disease. We focus on the chaperonins, which are essential for protein folding and salvage pathways in all three Domains of life. The eukaryotic CCT (chaperonin containing TCP1), a Group II chaperonin, is an obligate chaperone for at least 10% of eukaryotic proteomes [3,4]. Group II chaperonins, e.g. CCT, are found in Archaea and eukaryotes, and these consist of two stacked rings of eight non-identical subunits per ring
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