TY - JOUR
T1 - The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
AU - Campelo, Diana
AU - Lautier, Thomas
AU - Urban, Philippe
AU - Esteves, Francisco
AU - Bozonnet, Sophie
AU - Truan, Gilles
AU - Kranendonk, Michel
N1 - info:eu-repo/grantAgreement/FCT/3599-PPCDT/135794/PT#
This work was in part funded by a joint ANR/FCT program; France: ANR-13-ISV5-0001 (DODYCOEL), and Portuguese national funds, through the Fundacao para a Ciencia e a Tecnologia (Project FCT-ANR/BEX-BCM/0002/2013).
PY - 2017/10/30
Y1 - 2017/10/30
N2 - NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.
AB - NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.
KW - Conformational exchange
KW - Diflavin reductase
KW - Electron transfer
KW - Multidomain proteins
KW - Protein dynamics
KW - Protein-protein interaction
UR - http://www.scopus.com/inward/record.url?scp=85032575755&partnerID=8YFLogxK
U2 - 10.3389/fphar.2017.00755
DO - 10.3389/fphar.2017.00755
M3 - Article
C2 - 29163152
AN - SCOPUS:85032575755
SN - 1663-9812
VL - 8
SP - Online
JO - Frontiers in Pharmacology
JF - Frontiers in Pharmacology
IS - OCT
M1 - 755
ER -