Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data.
To identify the binding site for bovine cytochrome b(5) (cyt b(5)) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with (2)H- and (15)N-enriched cyt c and unlabeled cyt b(5). In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b(5) concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A K(a) of (4 +/- 3) x 10(5) M(-)(1) was obtained with a lower limit of 855 s(-)(1) for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b(5) heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c-cyt b(5) complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein-protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces.
Deep
Shashank
S
Biophysics Research Division, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, USA.
Im
Sang-Choul
SC
Zuiderweg
Erik R P
ER
Waskell
Lucy
L
D000818:Animals; D002247:Carbon Isotopes; D002417:Cattle; D015786:Cytochromes b5 / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006736:Horses; D009097:Multienzyme Complexes / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D013499:Surface Properties; D017186:Titrimetry
D002247:Carbon Isotopes; D009097:Multienzyme Complexes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D045304:Cytochromes c; D015786:Cytochromes b5