Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

Abstract

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with exchange between conformational states proposed to regulate changes in function. Here we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an "induced fit" mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

Structural Dynamics of the Precatalytic State of Human Cytochrome c upon T28C, G34C, and A50C Mutations: A Molecular Dynamics Simulation Perspective

“…In general, it is acceptable that the peroxidase activity of cytc is induced by the dissociation of Met80 and, therefore, enables the access of substrates to the pentacoordinated heme iron. However, the classical model of the hexacoordinate form is thus utilized in our MD study to investigate the conformational dynamics of the precatalytic structures of hCytc. ,, In this form, different structural dynamics are found in the G34C mutant, as suggested by rmsd, Rg, SASA, and rmsf calculations.…”

“…Additionally, other structural modifications of cytc, such as post-translational modification and mutations, culminate in alternative conformations that support the increment of peroxidase activity. These include the naturally occurring mutations of human cytc (hCytc) at positions G41S, Y48H, and A51V. , All are related to the pathophysiology of thrombocytopenia 4 (THC4), a disorder characterized by abnormally low counts of platelets. The findings have emphasized the important features of various ranges of conformational states of the protein that are closely relevant to biological functions.…”

Cytochrome c Interaction with Cardiolipin Plays a Key Role in Cell Apoptosis: Implications for Human Diseases

“…The protein exhibits fully reversible folding-unfolding; the foldon that forms first (i.e., the most stable) is the last to unfold while the foldon that forms last (i.e., the least stable) is the first to unfold. In cyt c the foldon that forms first (i.e., the most stable) is that formed by the N-and C-terminal helices, while the one that folds last (i.e., the least stable) is the foldon made up of the omega-loop C (residues [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. The stability of foldons is indicated as follows (colors refer to the regions illustrated in Figure 1B): red unit > grey unit > green unit > blue unit > yellow unit.…”

“…All of these mutations cause a rare autosomal dominant disorder, thrombocytopenia, along with a heterogeneous group of other inherited diseases. All are characterized by low platelet counts (less than 150,000 platelets/µL in the blood) and induce mitochondrial apoptosis [44,45].…”

Cytochrome c in cancer therapy and prognosis

“…Lysine mutants of cyt c have shown deficient apoptosome function, by inhibiting Apaf-1 oligomerization [ 66 ]. On the other hand, three cyt c pathogenic variants, Gly41Ser, Tyr41His, and Ala51Val, could increase the efficiency of apoptosome activation, possibly by enhancing cyt c flexibility and facilitating its interaction with Apaf-1 [ 67 ].…”