Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog

Abstract

The human chaperonin complex is a ~ 1 MDa 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.

Keywords: CCT5; Chaperonin; Chaperonopathies; DLS, dynamic light scattering; DSC, differential scanning calorimetry; ITC, isothermal titration calorimetry; Neuropathy; Pf, Pyrococcus furiosus; Pf-CD1, Pyrococcus furiosus chaperonin subunit with the last 22 amino acids deleted; Protein calorimetry; Pyrococcus furiosus.

Graphical abstract

Fig. 1

DSC. Thermograms for Pf-CD1 (red), Pf-H (green), and Pf-R (blue), all at 7 μM. The proteins show biphasic melting, but in Pf-CD1 the two peaks overlap, implying closer coupling between hexadecamer disassembly and denaturation of monomers. Calorimetric traces are given after subtraction of the instrumental base line. The scan rate was 60 °C/h.

Fig. 2

DSC at different protein concentrations: 7 μM (red), 3.5 μM (blue), and 1.7 μM (black). A, B, C, thermograms for Pf-CD1, Pf-H, and Pf-R, respectively. A1, B1, and C1, variations in total measured enthalpy as a function of concentration.

Fig. 3

ITC. A and B, ITC of the oligomer assembly process for Pf­ CD 1 (red), Pf-H (green), and Pf-R (blue). A, measured heat for 18 successive injections and best fit to a two-state curve according to a hexadecamer-monomer equilibrium. B, the corresponding normalized molar heat functions. C-E, ITC of ATP binding to the chaperonins. C (Pf-CDl), D (Pf-H), and E (Pf-R), enthalpies as a function of ATP stoichiometry, with the best-fit curves assuming a single binding site per subunit. A molar ratio of 16 corresponds to one ATP per subunit.

Fig. 4

Protein structure around the mutation site, showing the superposed crystal structures from Thermococcus (Tk, PDB:1Q3Q, green) and yeast (4V81, orange). The Thermocuccus and Pf sequences are >90% identical, so the green structure gives a reliable model for Pf. The yeast structure overlays closely, consistent with the high homology of all known CCT sequences, and enabling 4V81 here to represent the human structure. The mutation site (Ile-138 in both archaea, yellow) and its close neighbors, which are identical in Tk and Pf. The mutation site is just below the protein surface in a sterically restricted pocket in all the homologs. In human CCT5, the wild-type residue corresponding to Ile-138 is a His (cyan). The pathological mutation replaces His with Arg (red). Although His is accommodated in the normal human protein, the Arg side chain appears to be sterically and electrostatically unfavorable in both the archaeal and human frameworks. Image made using PyMOL Molecular Graphics System, Version 1.5, Schrodinger.