A model of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data

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.

Keywords: Cytochrome P450; Enzyme Catalysis; Membrane Proteins; NMR; Structural Biology.

FIGURE 1.

NMR structure of rabbit microsomal cytb₅. A, NMR structure of full-length cytb₅ obtained from a combined solution and solid-state NMR approach. The soluble heme domain structure (residues 1–104) of full-length cytb₅ was solved in DPC micelles by solution NMR, with a backbone r.m.s.d. of 0.32 ± 0.10 Å. The transmembrane domain structure (residues 106–126) of full-length cytb₅ was determined in aligned DMPC/DHPC bicelles using solid-state NMR spectroscopy. B, ¹H-¹⁵N TROSY-HSQC spectrum of uniformly, ¹³C-, ²H-, and ¹⁵N-labeled cytb₅ in micelles exhibiting well resolved peaks. C, two-dimensional HIMSELF spectrum of uniformly ¹⁵N-labeled cytb₅ reconstituted in aligned DMPC/DHPC bicelles. The blue ring presents the best fit for the helical wheel pattern of resonances from the α-helical transmembrane domain of cytb₅.

FIGURE 2.

High resolution solution NMR spectrum of cytb₅. A, ¹H-¹⁵N TROSY-HSQC spectrum of uniformly ¹³C-, ²H-, and ¹⁵N-labeled full-length mammalian cytb₅ in DPC micelles. The backbone resonance peaks are labeled with the residue-specific assignment of cytb₅. Unlabeled peaks include side chain resonances (Asn, Gln) and the lower populated cytb₅ isomer. Tryptophan indole protons between 10 and 11 ppm were not assigned due to broadening. B, expansion of the crowded region of the ¹H-¹⁵N TROSY-HSQC spectrum.

FIGURE 3.

Solution NMR structure of the cytosolic domain of full-length cytb₅ in DPC micelles. A, overlay of the 20 lowest energy structures of cytb₅ generated from CYANA2.1 based on the NMR restraints in Table 1. The backbone (Lys-7 to Arg-89) r.m.s.d. was 0.32 Å. B, two different views of the overlay of the 20 lowest energy structures of the heme domain of cytb₅ generated from HADDOCK after docking heme B. His-44 and His-68, which coordinate the heme, are represented as green sticks. C, three different views of the structure of the heme domain of cytb₅ obtained from solution NMR, with the heme B molecule orientation obtained from HADDOCK. Left, middle, and right orientations show the proximal, bottom (lower edge of the cleft), and side view of cytb₅, respectively.

FIGURE 4.

Transmembrane domain of cytb₅ is visible under magic angle spinning NMR. A, ¹H-¹⁵N-HMQC spectrum recorded on a selectively [¹⁵N]alanine-labeled sample of cytb₅ incorporated in DPC micelles. This spectrum was obtained from a 600 MHz Varian solid-state NMR spectrometer under a 2.5 kHz spinning speed of the sample at 37 °C, using a double-resonance magic angle spinning nanoprobe (Agilent/Varian). B, representation of cytb₅ highlighting all the alanines (red sphere) in the protein.

FIGURE 5.

Chemical shift perturbation analysis. A histogram presenting the experimentally measured changes in chemical shift values for residues in cytb₅ upon complex formation with cytP450. The change in the chemical shift was calculated using the standard formula Δδ = √{(ω_HNΔ¹HN)² + (ω_NΔ¹⁵N)²}, where ω_H = 1, ω_N = 0.154, and Δδ represents the average (NH) chemical shift perturbation (82). The chemical shift perturbations are represented as a continuous color map on the NMR structure of cytb₅. Resonances for His-32, Gly-46, His-68, and Ser-69 (represented in magenta) disappear upon complex formation.

FIGURE 6.

Mapping the effect of cytP450 binding to cytb₅ measured from NMR. A, histogram representing the differential line broadening NMR data for the cytb₅-cytP450 complex. The amide peak intensities for free cytb₅ are presented in red. Yellow presents the intensities for cytb₅ residues in a 1:1 equimolar complex with substrate-free cytP450. Green, cyan, and magenta highlight the extensive peak broadening observed for cytb₅ residues upon addition of the increasing amounts of unlabeled cytP450 bound to BHT (A = 1:0.3, B = 1:0.6, and C = 1:1 molar ratios between cytb₅ and cytP450). All peak intensities were normalized to the C-terminal residue Asp-134 in the unbound cytb₅ spectrum to account for the change in intensity upon complex formation. B and C present two different views of cytb₅ rotated by 90° with respect to each other and a space-filling representation of the second view. B, cytb₅ residues exhibiting extensive line broadening (with a decrease in peak height >20% as compared with free cytb₅) upon complex formation with an equimolar amount of substrate/ligand-free cytP450 are colored orange onto the NMR structure of cytb₅. C, residues of cytb₅ whose resonances are broadened beyond detection upon complex formation with an equimolar amount of cytP450 bound to BHT are represented in magenta. All NMR data were collected on the full-length complex incorporated in isotropic bicelles.

FIGURE 7.

Structure of the full-length membrane-bound cytb₅-cytP450 complex. A, two lowest energy clusters (I and II) of the complex between the catalytic heme-binding domains of rabbit cytb₅ (NMR structure; blue) and cytP4502B4 (PDB code 1SUO (41); in gold) generated from HADDOCK, driven by NMR, and mutagenesis restraints. Heme molecules are presented in red. B, proposed electron transfer pathway between the redox centers of cytb₅ and cytP450 are presented as broken lines. The shortest electron transfer pathway predicted using HARLEM (76) is shown in the black dotted lines for clusters I and II. The shortest heme-edge to heme-edge distance is 7.4 and 9.0 Å in clusters II and I, respectively.

FIGURE 8.

Binding interface of the membrane-bound cytb₅-cytP450 complex. The complex is presented by opening the complex-like pages of a book with the interaction interface of cytb₅ and cytP450 facing the viewer. The space-filling model of cytb₅ (NMR structure) and cytP450 (PDB code 1SUO (41)) is presented highlighting the interfacial residues involved in protein-protein contacts in cluster I (A) and cluster II (B) complex structures. Residues on cytb₅ that are in contact with residues on cytP450 are denoted with matching letters in parentheses. For example, Asp-65 (orange) on cytb₅ is H-bonding to Arg-122 (blue) on cytP450 in A. Arg-125 highlighted in blue is H-bonded to the heme-d-propionate in B. An important point to note is that the residues on cytb₅ and cytP450, which form the interaction interface, are largely the same between the two clusters and are mostly present on the lower edge (residues on α4 and α5 helix) of the soluble domain surrounding the heme. The residues in the interface are in excellent agreement with our NMR data and site-directed mutagenesis presented here (Tables 3 and 4), as well as elsewhere (15).