Identification of productive and futile encounters in an electron transfer protein complex

Abstract

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

Keywords: cytochrome P450cam; encounter complex; maximum occurrence; paramagnetic NMR; putidaredoxin.

Fig. 1.

Paramagnetic lanthanoid/MTSL tag positions and distribution of PREs unaccounted for by the main state of the complex. For each paramagnetic probe, the amide protons of the reporter protein with PREs unaccounted for by the main state are indicated by gray spheres—large spheres signify strongly misfitted PREs (used to construct the clusters), and smaller spheres signify moderately (∼20 s⁻¹) misfitted ones. (A) Probes placed on cytP450cam (PREs measured on Pdx). (B) Probes placed on Pdx (PREs measured on cytP450cam).

Fig. 2.

Observed and calculated PREs. The experimental PREs are shown as blue dots, the PREs back-calculated from the main state alone are shown as a red line, and those from an ensemble containing 99.4% of the main state complemented with 0.6% of other conformers optimized to fit the experimental data are shown as a green line. The complementing structures were selected from the broad conformational pool (in the text), and their specific weights were chosen through the ensemble optimization algorithm of the MaxOR/minOR program. The error bars represent propagated errors based on the noise level in the NMR spectra and the fitted error in the determination of the diamagnetic transverse relaxation rate. The errors for probes 4 and 6 are noticeably higher because of the lower intensity of the cytP450cam-detected spectra.

Fig. S1.

Clustering of residues for which PREs unaccounted for by the main state were measured. The residues in green are those for which PREs were not measured or were successfully fitted by the main state; other residues are colored according to the clusters to which they belong. The solid spheres of a given color depict the geometric center of the corresponding cluster. (A) Probe 2 (PREs measured on Pdx): cluster A, red; cluster B, blue; and cluster C, orange. (B) Probe 3 (PREs measured on Pdx): cluster D, red; cluster E, blue; and cluster F, orange. (C) Probe 4 (PREs measured on cytP450cam): cluster G, red; cluster H, blue; and cluster I, orange. (D) Probe 6 (PREs measured on cytP450cam): cluster J, red; cluster K, blue; and cluster L, orange. CytP450cam orientation is the same as in Fig. 1.

Fig. 3.

Representative minOR regions and their counterparts shrunk by assuming sparsity. (A) The smallest region with minOR > 0 for cluster 2.C; cytP450cam is shown as a green cartoon, with the positions of probes 1–3 indicated as black, orange, and yellow axis frames, respectively; the blue dots represent the centers of mass of Pdx in all of the conformations belonging to the region, and the gray sphere indicates the position of Pdx in the stereospecific complex. (B) The three regions with minOR > 0 for probe 2 [clusters A (red), B (orange), and C (blue)] represented in the Pdx frame of reference; Pdx is shown in green cartoon, and the blue dots mark the positions of probe 2 (on cytP450cam) in all of the conformations composing the regions. (C) The subsections of the region shown in A (cluster 2.C) obtained by applying an assumption of sparsity (in the text): each sphere marks the center of a 5 × 5 × 5-Å cube, and the different shades of blue denote the quality of the fit [from dark blue (Q = 0) to light blue (Q = 0.2)]. (D) Region 6.L before (Left) and after (Right) applying the assumption of sparsity (represented as in A and C, respectively).

Fig. S2.

The smallest regions with minOR > 0 (Left) and all of the 5 × 5 × 5-Å subsections of these regions containing structures that fit all of the PREs in the given cluster simultaneously to a Q < 0.2 (Right). The cytP450cam is shown as green cartoon, with the positions of probes 1–3 indicated as black, orange, and yellow axis frames, respectively. The blue dots represent either the centers of mass of Pdx (Left) or the 5 × 5 × 5-Å subregions (Right). The different shades of blue in Right denote the quality of the fit [from dark blue (Q = 0) to light blue (Q = 0.2); in the text]. The letter codes A–L are used to label the regions obtained through a clustering procedure (see main text).

Fig. 4.

The positive (blue) and negative (red) electrostatic potential isosurfaces [obtained using APBS (61) and traced in PyMol (62) at 0.7 kT/e] of cytP450cam are depicted together with the center of mass positions of Pdx in the encounter regions obtained after applying the scarcity assumption (see text) shown as a mesh, with the colors representing the states related to the different probes; probe 2, orange; probe 3, yellow; probe 4, cyan; probe 6, magenta. The colored spheres are representatives of the respective encounter regions placed above positively charged surface areas. The gray sphere represents the main state. The green bars indicate the probe positions on cytP450cam. The curved arrows indicate possible paths between the minor states and the main binding site, suggesting that these states could represent productive encounter sites. Regions 4.G and 6.J illustrate isolated states that may represent futile encounter sites.