Increased dynamics in the 40–57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

Karsisiotis, Andreas Ioannis; Deacon, Oliver M.; Wilson, Michael T.; Macdonald, Colin; Blumenschein, Tharin M. A.; Moore, Geoffrey R.; Worrall, J.A.R.

doi:10.1038/srep30447

Cited by 56 publications

(142 citation statements)

References 57 publications

(99 reference statements)

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“…The physical and biochemical properties of the G41S, G41A and G41T variants have been extensively studied. 25, 60–64 All three variants show increased peroxidase activity 61 that correlates with the stability of the heme crevice as measured by the accessibility of the alkaline conformer and the strength of the heme-Met80 bond. 62 However, introduction of the G41S mutation into mouse Cyt c shows that the correlation between a more accessible alkaline state and higher peroxidase activity is not absolute, 61 consistent with our results for K72A Hu Cyt c .…”

Section: Discussionmentioning

confidence: 98%

“…HD exchange and backbone dynamics measured by NMR show that the G41S mutation in Hu Cyt c enhances the dynamics of both the 40–57 and 70–85 Ω-loops of Hu Cyt c , suggesting that increased dynamics near the heme are primarily responsible for the enhance peroxidase activity. 64 As noted in the studies on the peroxidase activity of G41S Cyt c , the effects of the intrinsic peroxidase activity of Cyt c when it is not bound to cardiolipin remain unclear. 61 However, the much lower intrinsic peroxidase activity of Cyt c from higher eukaryotes, like horse 12, 34 and human, compared to lower eukaryotes like yeast, 12 show that Cyt c has evolved to limit intrinsic peroxidase activity.…”

Section: Discussionmentioning

confidence: 99%

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Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

et al. 2017

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We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland et al. PNAS 111, 6648–6653 (2014)]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pKa of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 units. Consistent with the increase in the apparent pKa, the rate of formation of the dominant alkaline conformer decreases and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases kcat by 1.6– to 4–fold at pH values from 7 to 10, a larger effect than for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the larger effect of the K72A mutation on peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human versus Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

et al. 2017

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“…Remarkably, all the tested alterations of Ser47 resulted in an inefficient caspase activator . Moreover, the G41S and Y48H mutants, which are responsible for the onset of the thrombocytopenia‐4 disease, showed enhanced caspase‐3 activation activity .…”

Section: Cytochrome C In the Cytoplasm: Established And New Targetsmentioning

confidence: 96%

New moonlighting functions of mitochondrial cytochrome c in the cytoplasm and nucleus

et al. 2019

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Cytochrome c (Cc) is a protein that functions as an electron carrier in the mitochondrial respiratory chain. However, Cc has moonlighting roles outside mitochondria driving the transition of apoptotic cells from life to death. When living cells are damaged, Cc escapes its natural mitochondrial environment and, once in the cytosol, it binds other proteins to form a complex named the apoptosome—a platform that triggers caspase activation and further leads to controlled cell dismantlement. Early released Cc also binds to inositol 1,4,5‐triphosphate receptors on the ER membrane, which stimulates further massive Cc release from mitochondria. Besides the well‐characterized binding proteins contributing to the proapoptotic functions of Cc, many novel protein targets have been recently described. Among them, histone chaperones were identified as key partners of Cc following DNA breaks, indicating that Cc might modulate chromatin dynamics through competitive binding to histone chaperones. In this article, we review the ample set of recently discovered antiapoptotic proteins—involved in DNA damage, transcription, and energetic metabolism—reported to interact with Cc in the cytoplasm and even the nucleus upon DNA breaks.

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“…S5A). An increased dynamics in the Ω NY -loop has recently been observed in the G41S variant of Cc (42). The Ω NY -loop (also known as foldon V) and helix α 2 constitute the less-stable folding unit of Cc (43).…”

mentioning

confidence: 99%

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Moreno‐Beltrán

Guerra‐Castellano

Díaz-Quintana

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

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Increased dynamics in the 40–57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

Cited by 56 publications

References 57 publications

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

New moonlighting functions of mitochondrial cytochrome c in the cytoplasm and nucleus

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

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