Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Abstract

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations.

Fig. 1

Specific form of Cc–CcP complex. a Crystallographic Cc–CcP binding orientation (Pelletier and Kraut 1992). Cc and CcP are in grey and yellow, with heme groups in sticks. Cα atoms of CcP residues used for spin-labeling are shown as spheres, colored according to whether the attached SL exhibits intermolecular PREs (red) or not (blue). One SL position (K97, blue) is not seen in this view. b Definition of the spherical coordinates used in this work. Proteins’ centers of mass are shown as grey spheres, with CcP at the origin of the coordinate system. Cc possesses three translational (θ, φ, r) and three rotational (χ, ψ, ξ) degrees of freedom. For the definition of the rotational axes see “Materials and methods”. All protein representations in this work are visualized with PyMOL (DeLano 2002)

Fig. 2

Spatiotemporal analysis of Cc–CcP encounter state. a Interaction grid isosurface θ, φ, r _(χ,ψ,ξ=0) consisting of 12,205 Cc CMs (blue dots). b Isosurface in (a) coloured according to p _max(θ, φ) (Eq. 4), ranging from 0 (blue) to 0.3 (red). The white curve limits an area around the dominant form of the complex that contains Cc orientations contributing ≥5 Hz to . CcP (grey surface) and Cc (cartoon) in the top panels are in the same orientation as in Fig. 1a. The middle and bottom views are obtained by 180° rotation of the top-panel representations around, respectively, z and y axes. For each SL the oxygen atoms of 150 conformers used for ensemble averaging are space filled and colored red and blue to indicate, respectively, the presence and absence of the measured paramagnetic effects. The cyan sphere shows Cc CM in the dominant complex

Fig. 3

Experimental coverage of the conformational space of Cc–CcP encounter state. Cc CMs for the orientations that, respectively, violate experimental PREs (blue) or contribute ≥5 Hz to (red) at a p = 0.01 and b p = 0.1. Combination of the blue and red areas defines the total conformational space covered by the effects from the introduced SLs. The same isosurface as in Fig. 2a, b is shown. See the legend to Fig. 2 for more details. c Plot of experimental coverage, or surface area of CcP covered by PREs from a single SL, versus the population of the minor species

Fig. 4

PRE-based ensemble simulations of the Cc–CcP encounter state. a Intermolecular Q factors: Q _e (black), Q _ee (red), and Q _free (blue). See text for the definitions. b Correlation between the observed and calculated PREs for the dominant form of the complex alone (N = 0, top) or in combination with the simulated encounter ensemble (N = 10, bottom). c Observed and calculated PREs for Cc–CcP-SL complexes, with SLs attached at position N38C (top), N200C (middle) and T288C (bottom). Experimental (black; Volkov et al. ; Bashir et al. 2010), for the specific orientation (blue), and for the combination of the specific form and an encounter ensemble (N = 10, red). Crosses indicate the value of for the calculated PREs or identify the residues whose resonances disappear in the paramagnetic spectrum. The errors are standard deviations

Fig. 5

Spatial distribution of the encounter ensembles. a Reweighted atomic probability density maps for the overall distribution of Cc molecules obtained from 100 PRE-based ensemble calculations (N = 10, plotted at a threshold of 20% maximum). In the bottom view, SL atoms are removed for clarity. b Overlay of the Cc–CcP interaction isosurface coloured according to the experimental PREs at p = 0.03 (see the legend to Fig. 3 for details) and CMs of Cc molecules from 100 PRE-based ensemble simulations (N = 10, green spheres)

Fig. 6

Comparison of the theoretical and experimental simulations of the Cc–CcP encounter state. a Crystallographic Cc–CcP orientation. To facilitate the comparison with the published data, three aspartates of CcP are labeled and shown as orange sticks. b Overall distribution of 1,701 Cc molecules in the simulated Cc–CcP encounter complex (Bashir et al. 2010), displayed as a reweighted atomic probability density map (Schwieters and Clore ; plotted at a threshold of 0.5% maximum, blue). The surfaces of a Cc and b CcP are colored by the electrostatic potential calculated at ±5 k _B T (red—negative, blue—positive) with APBS (Baker et al. 2001). c The Boltzmann-averaged total electrostatic potential energy of interaction between CcP and horse Cc in units of k _B T as a function of Cc CM. This panel is taken from ref. (Northrup et al. 1988) with permission from Science. d The blue and green meshes indicate reweighted atomic probability density maps for the overall distribution of Cc molecules obtained from, respectively, Monte-Carlo simulations (same as in b) and PRE-based ensemble calculations (same as in Fig. 5a), both plotted at a threshold of 20% maximum. The dominant form of the complex is in the same orientation as in Fig. 1a. In the bottom panel, SL atoms are removed for clarity