Insights into the role of substrates on the interaction between cytochrome b5 and cytochrome P450 2B4 by NMR.

Meng Zhang,Stéphanie V Le Clair,Lucy Waskell,Sang-Choul Im,Ayyalusamy Ramamoorthy,Rui Huang,Shivani Ahuja

doi:10.1038/srep08392

Meng Zhang, Stéphanie V Le Clair + Show 5 more

Open Access

https://doi.org/10.1038/srep08392

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Abstract

Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.

Highlights

Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR
Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process
Through the use of Circular Dichroism (CD) experiments, in which NaCl was titrated into cytochrome P450 2B4 (CYP2B4) incorporated in isotropic bicelles composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) (q 5 0.25), we confirm that the overall secondary structure of CYP2B4 does not change with the addition of NaCl (Fig. S1)

Summary

Introduction

Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process. A more detailed look into the cyt b5-P450 complex formation process under the influence of substrates, in a native-like environment, will lead to a better understanding of the role that substrates play in modulating the interactions between these two redox partners. NMR techniques are used to investigate the interactions, at the atomic level, between full-length substrate-free/BHTbound CYP2B4 and full-length rabbit cyt b5 in a membrane mimetic environment under different ionic strength conditions. A detailed analysis of the differential ionic strength dependence of resonance intensities of cyt b5 residues allowed for identification of residues most likely involved in specific interactions with CYP2B4, leading to a better understanding of the complex interface

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