A tale of two charges: Distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c

2001 ◽  
Vol 79 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Jonathan C Parrish ◽  
J Guy Guillemette ◽  
Carmichael JA Wallace
Keyword(s):  
Cytochrome C ◽  
Basic Amino Acid ◽  
Intermembrane Space ◽  
Intrinsic Structure ◽  
Conserved Residues ◽  
Dynamics Simulations ◽  
And Function ◽  
Saccaromyces Cerevisiae ◽  
Electron Transport Protein ◽  
Horse Cytochrome

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electro statically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.Key words: cytochrome c, structure-function studies, molecular modelling, surface electrostatics.

scholarly journals Preservation of Mitochondrial Structure and Function after Bid- or Bax-Mediated Cytochrome c Release

2000 ◽  
Vol 150 (5) ◽  
pp. 1027-1036 ◽  
Author(s):  
Oliver von Ahsen ◽  
Christian Renken ◽  
Guy Perkins ◽  
Ruth M. Kluck ◽  
Ella Bossy-Wetzel ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
And Function

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1–mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

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

2021 ◽  
Vol 478 (3) ◽  
pp. 669-684
Author(s):  
Matthias Fellner ◽  
Rinky Parakra ◽  
Kirstin O. McDonald ◽  
Itamar Kass ◽  
Guy N.L. Jameson ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cytochrome C ◽  
Peroxidase Activity ◽  
Conformational Dynamics ◽  
Cellular Processes ◽  
Pathogenic Variants ◽  
Alkaline Transition ◽  
Apoptotic Activity ◽  
Dynamics Simulations ◽  
And Function

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 an 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 the 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.

scholarly journals Effects of Liposome and Cardiolipin on Folding and Function of Mitochondrial Erv1

2020 ◽  
Vol 21 (24) ◽  
pp. 9402
Author(s):  
Xiaofan Tang ◽  
Lynda K Harris ◽  
Hui Lu
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Protein Import ◽  
Reductase Activity ◽  
Catalytic Efficiency ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Cytochrome C Reductase ◽  
Mitochondrial Intermembrane Space ◽  
And Function

Erv1 (EC number 1.8.3.2) is an essential mitochondrial enzyme catalyzing protein import and oxidative folding in the mitochondrial intermembrane space. Erv1 has both oxidase and cytochrome c reductase activities. While both Erv1 and cytochrome c were reported to be membrane associated in mitochondria, it is unknown how the mitochondrial membrane environment may affect the function of Erv1. Here, in this study, we used liposomes to mimic the mitochondrial membrane and investigated the effect of liposomes and cardiolipin on the folding and function of yeast Erv1. Enzyme kinetics of both the oxidase and cytochrome c reductase activity of Erv1 were studied using oxygen consumption analysis and spectroscopic methods. Our results showed that the presence of liposomes has mild impacts on Erv1 oxidase activity, but significantly inhibited the catalytic efficiency of Erv1 cytochrome c reductase activity in a cardiolipin-dependent manner. Taken together, the results of this study provide important insights into the function of Erv1 in the mitochondria, suggesting that molecular oxygen is a better substrate than cytochrome c for Erv1 in the yeast mitochondria.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

scholarly journals Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

2021 ◽  
Vol 11 (9) ◽  
pp. 4048
Author(s):  
Javier A. Linares-Pastén ◽  
Lilja Björk Jonsdottir ◽  
Gudmundur O. Hreggvidsson ◽  
Olafur H. Fridjonsson ◽  
Hildegard Watzlawick ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Ligand Docking ◽  
Glycoside Hydrolase Family ◽  
Ligand Interactions ◽  
Tim Barrel ◽  
Branching Point ◽  
The Difference ◽  
Dynamics Simulations ◽  
And Function ◽  
Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

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