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The orientations of cytochrome c in the highly dynamic complex with cytochrome b₅ visualized by NMR and docking using HADDOCK

¹ Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands
² Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands

(RECEIVED September 30, 2004; FINAL REVISION September 30, 2004; ACCEPTED November 19, 2004)

The interaction of bovine microsomal ferricytochrome b₅ with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for ¹H and ¹⁵N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein–protein docking simulations have been performed for the binary cytochrome b₅–cytochrome c and ternary (cytochrome b₅)–(cytochrome c)₂ complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b₅. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

Keywords: cytochrome c; cytochrome b₅; docking; HADDOCK; NMR; electron transfer

Abbreviations: AIR, ambiguity-driven intermolecular restraints • cyt b₅, cytochrome b₅ • cyt c, cytochrome c • EM, energy minimization • ET, electron transfer • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • Pr, heme propionate • RMSD, root-mean-square deviation • SA, simulated annealing • 1D, one-dimensional • 2D, two-dimensional

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041150205.

Reprint requests to: Marcellus Ubbink, Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands; e-mail: m.ubbink{at}chem.leidenuniv.nl; fax: +31-71-527-4349.

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