Altered conformational dynamics contribute to species-specific effects of cytochrome c mutations on caspase activation

Abstract

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

Keywords: Apaf-1; Apoptosis; Cytochrome c; Molecular dynamics; Peroxidase.

Fig. 1

Structural alignment of mouse and human cytochromes c. Mouse (cyan) and human (grey) cytochrome c structures are superimposed with the 40–57 Ω-loops coloured in orange (mouse) and yellow (human). The heme is coloured red (mouse) and grey (human). Residue differences between structures shown as sticks labelled with three-letter residue codes (black for human, blue for mouse) and residue position number. Heme propionates (hp) 6 and 7 and the position of Gly41 are also shown. Drawn from PDB entries 3ZCF (human) and 5C0Z (rat, which has an identical sequence to mouse)

Fig. 2

Mainchain movement of mouse and human cytochrome c variants. Root mean squared deviation (RMSD) (Å) between the 5C0Z (mouse) or 3NWV (human) starting structure and the states captured during the MD simulations for each cytochrome c variant. A Mouse WT cytochrome c runs. B Mouse G41S cytochrome c runs. C Human WT cytochrome c runs. D Human G41S cytochrome c runs. FeII_1 runs are shown in blue, FeII_2 in cyan, FeIII_1 in dark red, FeIII_2 in orange

Fig. 3

Gibbs–Helmholtz plot of mouse and human WT and G41S cytochromes c. Gibbs–Helmholtz plot calculated with the unfolding transition data for msWT (green), msG41S (blue), huWT (black) and huG41S (orange) from Table 2. The dashed lines in matching colour represent the errors reported in Table 2. The Gibbs–Helmholtz plot is defined by the equation: ∆G = ∆H(1-T/T_m). Dashed line at ∆G = 0, where unfolding/refolding is neither favourable nor unfavourable

Fig. 4

Selected residue distances for HuG41S FeII_1 simulation. Distance (Å) over huG41S FeIII_1 simulation for Asn52δO to Thr78OG1 (blue) and Thr78γO to the closest oxygen of hp6 (pink)