NMR studies on holo-CcmE and in vivo mutagenesis studies on the interaction between CcmC and CcmE

At least five different systems are responsible for the maturation of c-type cytochromes. System I, present in the mitochondria of photosynthetic organisms and most Gram-negative bacteria, is the most complex cytochrome c biogenesis system discovered. In the model organism Escherichia coli, more than 10 gene products work together to attach heme to the highly conserved CXXCH motif of the apo-cytochrome polypeptide. This system consists of proteins that chaperone the heme and the apo-cytochrome, and they ensure the correct assembly of the holo-cytochrome. In this thesis, CcmC and CcmE, two key players in the heme delivery part of System I prior to covalent attachment, have been investigated. Particular emphasis has been given to CcmE, an unusual heme chaperone that binds its heme via a covalent yet transient bond using its H130 residue.
Bioinformatics techniques have been used to identify potential key residues on CcmC and CcmE, especially residues with high conservation and/or covariance between the two proteins. Site-directed mutagenesis studies and in vivo experiments were used to demonstrate that three pairs of conserved polar amino acids sharing a common orientation on CcmC and CcmE are crucial for the assembly of the CcmC:heme:CcmE complex, an essential intermediate for holo-CcmE formation. Single and multiple variants of these polar amino acid pairs demonstrated that these residues drive the interaction between CcmC and CcmE. Covariance analysis identified two highly co-varying residues on CcmC and CcmE. It was demonstrated that these residues play an important role in fine-tuning the positioning of CcmE in its complex with heme-bound CcmC, and their relative size is crucial for their role. Any perturbations decreasing the size of these residues led to incomplete processing of holo-CcmE, and abolishment of cytochrome c maturation.
Holo-CcmE was reconstituted in vitro, and this protein was studied using 2D 1H- 15N HSQC. These studies provided residue-specific-level details on how the heme moiety interacts with the polypeptide in the covalently formed holo-CcmE. Contradictory to previous predictions, it was demonstrated that the heme moiety is not in close proximity to the core β-barrel fold of the protein. Rather, it was shown that heme interacts directly with the C-terminus. 2D 1H- 1H TOCSY studies were used to show that no tyrosine or phenylalanine ligands exist to the heme in holo-CcmE formed in vitro, suggesting that the protein most likely does not pack around the heme. These findings are consistent with the chaperone role of the protein, as the interaction of heme with the C-terminus enables its swift sequential transfer to the apo-cytochrome through CcmF.
Heme titrations probed via 2D 1H- 15N HSQC were carried out on the H130A variant of CcmE, which cannot bind heme covalently. These studies provided clear insight into the non-covalent interactions between CcmE and heme, and the putative heme pocket of the CcmE protein. It was demonstrated that no heme pocket exists on apo-CcmE, and any non-covalent interactions between CcmE and heme are located around the C-terminus, specifically around R148 and R149. 1H- 1H 2D TOCSY identified Y154 as a potential ligand of the non-covalently bound heme. It was demonstrated that the highly conserved Y134 residue acts during initial non-covalent interactions with heme, and then may ligand switch to the Y154 residue.

Advisor: Ferguson, S
Redfield, C