Abstract
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.
Highlights
Background: cytb5 modulates catalysis performed by cytsP450, in vivo and in vitro
To identify cytb5 residues involved in complex formation with cytP4502B4, we first solved the three-dimensional structure of ferric cytb5 in DPC micelles, using a combination of high resolution solution and solid-state NMR experiments (Fig. 1)
Here, we have presented the first full-length structure of rabbit cytb5 incorporated in a membrane mimetic (DPC micelles or lipid bicelles) obtained using a combination of solution and solid-state NMR spectroscopy
Summary
Background: cytb modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb was solved by NMR, and the cytP450-binding site on cytb was identified by mutagenesis and NMR. Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Model Complex between Mammalian cytb and Cytochrome P450 electron due to its high redox potential as compared with ferric cytP450 [7,8,9,10]. Cytb may enhance the rate of catalysis by up to 100-fold, whereas at high concentrations it inhibits catalysis by competing with CPR for a binding site on cytP450, thereby preventing the transfer of the first electron and the reduction of ferric cytP450 to the ferrous form [6, 7, 14]. When the stimulatory and inhibitory effects of cytb are equal, cytb will appear to have no effect on the catalytic activity of cytP450
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.