Michael Waterman
Faculty Member
Last active: 2/12/2015

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points.

Anandatheerthavarada HK, Amuthan G, Biswas G, Robin MA, Murali R, Waterman MR, Avadhani NG
EMBO J. 2001 20 (10): 2394-403

PMID: 11350928 · PMCID: PMC125462 · DOI:10.1093/emboj/20.10.2394

We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.

MeSH Terms (17)

Adrenodoxin Animals Binding Sites Cattle Chromosome Mapping Cross-Linking Reagents Cytochrome P-450 CYP1A1 Evolution, Molecular Flavodoxin Mammals Mitochondria, Liver Models, Molecular Mutagenesis NADPH-Ferrihemoprotein Reductase Protein Structure, Secondary Rats Two-Hybrid System Techniques

Connections (1)

This publication is referenced by other Labnodes entities:

Links