Structural differences between soluble and membrane bound cytochrome P450s

Abstract

The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F'- and G'-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Fugure 1

The initial state for molecular dynamics simulation of CYP3A4 in solution (A) and in the membrane bilayer (B). The protein is shown in green with the space filling heme colored orange and is inserted into the POPC bilayer consisting of 304 lipid molecules, hydrated with a 10 Ǻ solvent layer present at the borders of the box. Na⁺ and Cl⁻ ions are shown as yellow and green spheres.

Figure 2

The overall cytochrome P450 fold shown in comparing of structures of soluble CYP101 (A, C) and membrane bound CYP2E1 (B, D). A and B are a distal view while C and D are a heme edge view with the B-C helices and connecting loops removed to show the substrate binding pocket and the heme binding residue (blue sticks) hydrogen bonded to the A-propionate side chain. The beta-domain is shown in yellow, F-G helices in magenta, C-terminal beta-loop in orange and B-C loop in light blue. The heme is shown in red sticks. The beta-domain in CYP2E1 is significantly lower than in CYP101, opening the channel 2b over the A-propionate. The longer F-G loop in CYP2E1, with extra F’-helix, shifts the main access channel from 2a in CYP101 to 2b in CYP2E1

Figure 3

Angle between the A-propionate side chain (Cα – Cβ bond) and the mean heme plane (A) and the distance between the center of the beta-domain and the mean heme plane (B) are plotted against the number of the heme binding residue (HBR). Each cytochrome P450 is represented as one point, with the data averaged over multiple structures when available. Soluble enzymes from prokaryotes are shown as red circles, membrane bound eukaryotic enzymes as blue triangles. The dotted lines show separation of all proteins into two groups. For one, which includes almost all membrane cytochromes P450 except CYP51, the A-propionate side chain is directed towards the proximal side of the heme, and the center of the beta-domain is within 1.5- 4.2 Ǻ from the heme plane. For the second group the beta-domain is shifted towards the distal side of the heme plane by 4.2 – 9.0 Ǻ, and the A-propionate side chain points in the same direction in most of them. Exceptions labeled in both panels are from Streptomyces (CYP158, CYP154, CYP151, CYP170), Mycobacteria (CYP124, CYP125, CYP142), Bacillus subtilis (CYP134) and Pseudomonas (CYP108). Separation of cytochromes P450 on the molecular mass scale indicates the difference between smaller soluble and larger membrane bound enzymes. The latter have an extra N-terminal transmembrane fragment and several longer loops, including F-F’-G’-G helices suggested to be involved in interactions with the membrane.

Figure 4

Distribution of cytochromes P450 with respect to the height of the beta-domain over the heme plane for the 43 soluble cytochromes P450 (A), and 22 membrane bound enzymes (B). Heights have been calculated as described in Materials and Methods and averaged over multiple structures when available, to give one data point for each cytochrome P450. For 21 cytochromes (including 18 membrane bound) heights are in the 1.5 - 4.2 Ǻ range, 44 cytochromes have heights in the range 4.2 - 8.7 Ǻ (including four membrane bound eukaryotic cytochromes CYP51). The data used this figure are presented in the Table S1 in Supplemental Materials.

Figure 5

Substrate access pathways 2b (orange) and 2c (gray) calculated by CAVER for CYP3A4 incorporated into the membrane and in solution, (A) starting point for both MD simulations, (B) after 40 ns in aqueous solution, and (C) after 40 ns in the membrane environment. Both (B) and (C) remain stable for the remainder of the 50 ns MD simulation.