Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.

Original publication

DOI

10.1074/jbc.RA118.005024

Type

Journal article

Journal

J Biol Chem

Publication Date

26/10/2018

Volume

293

Pages

16778 - 16790

Keywords

CcmC, CcmE, Gram-negative bacteria, System I, cytochrome c, cytochrome c maturation, heme, nuclear magnetic resonance (NMR), post-translational modification (PTM), protein-protein interactions, Amino Acid Substitution, Apoproteins, Bacterial Outer Membrane Proteins, Binding Sites, Crystallography, X-Ray, Cytochromes c, Escherichia coli, Escherichia coli Proteins, Heme, Hemeproteins, Membrane Proteins, Mutagenesis, Site-Directed, Protein Conformation, Protein Interaction Domains and Motifs