Structural characterization of a flexible two-domain protein in solution using small angle X-ray scattering and NMR data

Abstract

Multidomain proteins in which individual domains are connected by linkers often possess inherent interdomain flexibility that significantly complicates their structural characterization in solution using either nuclear magnetic resonance (NMR) spectroscopy or small-angle X-ray scattering (SAXS) alone. Here, we report a protocol for joint refinement of flexible multidomain protein structures against NMR distance and angular restraints, residual dipolar couplings, and SAXS data. The protocol is based on the ensemble optimization method principle (Bernadó et al., 2007) and is compared with different refinement strategies for the structural characterization of the flexible two-domain protein sf3636 from Shigella flexneri 2a. The results of our refinement suggest the existence of a dominant population of configurational states in solution possessing an overall elongated shape and restricted relative twisting of the two domains.

Figure 1

Structure of sf3636 and global variables describing the relative configuration of the two domains. The center of mass of the N-terminal (NTD) and C-terminal (CTD) domains, r_NTD and r_CTD, respectively, are shown as blue spheres. a) Ribbon diagram of a representative structure of the SCP ensemble. b) Relative domain position is described by three spherical coordinates (R_NC, θ, φ) that specify the relative position of the CTD center-of-mass r_CTD in the spherical coordinate system defined by the principal axes i_x,i_y, and i_z of the NTD inertia tensor. R_NC is the distance between positions of NTD and CTD centers-of-mass. c) Relative domains orientation is described by three anglesζ₁, ζ₂, and Ω. ζ₁ is a bond angle formed by vectors R_NC and u_N, where R_NC = r_CTD − r_NTD, and u_N is a vector connecting positions of Cα atoms of residues Leu16 and Lys42. ζ₂ is a bond angle formed by vectors R_NC and u_C, where u_C is a vector connecting positions of Cα atoms of residues Thr113 and Phe115. Ω is a torsion angle formed by vectors u_N, R_NC, and u_C. See also Figure S1.

Figure 2

Comparison of experimental (black circles) with predicted SAXS data. Prediction was made by ensemble fitting of SCP (red lines), NMR1 (cyan lines), and NMR2 (green lines) structural ensembles. See Table 2 and text for the ensemble definition. A) Scattering intensity plot. B) Kratky plot. C) Pair distance distribution function P(r).

Figure 3

Comparison of relative domain configuration in the structures of three different ensembles obtained using SCP approach. The backbone trace of the superimposed structures comprising the SCP (data in red), NMR (data in cyan), and NMR+RDC (data in green) ensembles are shown in E, F, and G, respectively. See Table 2 and text for the ensembles definition. The ensembles are overlaid with the ab-initio SAXS-predicted envelope and shown from two points of view. The right and left views are related by a 90° rotation. A) Distribution of the distance between NTD and CTD centers-of mass. B) Distribution of the inter-domain twist angle Ω (see Figure 1C). C) The spherical angles θ and φ specifying the relative domain position (see Figure 1B). D) The backbone trace of the 60 structures comprising the NMR (shown in cyan), NMR_+RDC (shown in green), and SCP (shown in red) ensembles. The N-terminal domains of all shown structures are superimposed. Two vectors, vector R _NC and vector of the unique principal axes of NTD, respectively, that form spherical angle θ are shown schematically as solid and dashed gray lines, respectively.

Figure 4

Experimental and calculated relaxation data for sf3636 at 11.74 Tesla. The experimental values (black bars) of amide T₁, T₂, and NOE versus residue number are shown in A, B, and C, respectively. The orange lines show the values obtained by averaging the experimental data over all residues from the secondary structure elements. The theoretical values of T₁ and T₂ were calculated for each SCP structure using the program HYDRONMR with the parameter a set to 2.2 Ǻ. The ensemble-averaged values of T₁ and T₂ are shown as blue filled circles. The horizontal bars on the top of each graph indicate the position of the secondary structure elements in the protein sequence. See also Figure S2 and Tables S1 and S2.

Figure 5

Effect of anisotropy on T₁/T₂ ratio for sf3636. A) The angle α (black bars) between N-H bond vector and the unique principal axis of the diffusion tensor as a function of residue number is calculated for a representative structure of the SCP ensemble. The domain-averaged values are shown as orange lines. B) The experimental (filled circles) and predicted (blue line) T₁/T₂ ratio as a function of the angle α. The angle α and predicted T₁/T₂ values were calculated using the rotational diffusion tensor that was obtained by fitting the structure to the experimental data and assuming rigid body tumbling of the protein molecule. The program MODELFREE was used to calculate the rotational diffusion tensor.

Figure 6

Comparison of relative domain configuration in the structures of three different ensembles obtained using EOM approach. The backbone trace of the superimposed structures comprising the OEP1, OEP2, and OEP12 ensembles is shown in A, B, and C, respectively. See Table 2 and text for the ensembles definition. The ensembles are overlaid with the ab-initio SAXS-predicted envelope (gray mesh) and shown from two points of view. The right and left views are related by a 90° rotation. D) The positions of CTD centers-of-mass in all structures from the OEP1 (blue spheres), OEP2 (magenta spheres), and OEP12 (brown spheres) ensembles with superimposed N-terminal domains (residues 8–55). For clarity, the structure of superimposed NTDs is shown as a ribbon diagram. Distribution of inter-domain distance R_NC in the OEP1 (E, blue), OEP2 (G, magenta) and OEP12 (F, brown) ensembles are shown as bars. The distribution of the interdomain twist angle Ω in the OEP1 (H, blue), OEP2 (J, magenta) and OEP12 (I, brown) ensembles are shown as bars. For comparison, the distributions in the NOE-restricted (E, H, black) and in the random (F, G, I, J, orange) pools of structures are shown as solid lines. See also Figures S3 and S4.

Figure 7

Comparison of the goodness-of-fit to the SAXS data for different structural models of sf3636 with their radius of gyration R_g (A) and maximum inter-atomic distance D_max (B). The goodness-of-fit to the SAXS data is measured by discrepancy χ. The data are shown for all structures in the random pool (orange dots) and in the SCP (red filled circles), OEP1 (blue filled circles), OEP2 (magenta filled circles), OEP12 (black filled circles), NMR (cyan filled circles), and NMR+RDC (green filled circles) ensembles. See Table 2 and text for the ensembles definition. χ-values for each structure were calculated using program CRYSOL. See also Figure