1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase.
The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15N and 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.
Barsukov
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I
Department of Biochemistry, University of Leicester, U.K.
Modi
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S
Lian
L Y
LY
Sze
K H
KH
Paine
M J
MJ
Wolf
C R
CR
Roberts
G C
GC
D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003198:Computer Simulation; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin / Q000201:enzymology
D002250:Carbon Radioisotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D006859:Hydrogen; D009251:NADPH-Ferrihemoprotein Reductase