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. 2018 Jul 17;115(2):289-299.
doi: 10.1016/j.bpj.2018.03.040.

Evaluating Anti-CD32b F(ab) Conformation Using Molecular Dynamics and Small-Angle X-Ray Scattering

Emma J Sutton  1 , Richard T Bradshaw  2 , Christian M Orr  3 , Bjorn Frendéus  4 , Gunilla Larsson  4 , Ingrid Teige  4 , Mark S Cragg  5 , Ivo Tews  6 , Jonathan W Essex  7
Affiliations

Evaluating Anti-CD32b F(ab) Conformation Using Molecular Dynamics and Small-Angle X-Ray Scattering

Emma J Sutton et al. Biophys J. .

Abstract

Complementary strategies of small-angle x-ray scattering (SAXS) and crystallographic analysis are often used to determine atomistic three-dimensional models of macromolecules and their variability in solution. This combination of techniques is particularly valuable when applied to macromolecular complexes to detect changes within the individual binding partners. Here, we determine the x-ray crystallographic structure of a F(ab) fragment in complex with CD32b, the only inhibitory Fc-γ receptor in humans, and compare the structure of the F(ab) from the crystal complex to SAXS data for the F(ab) alone in solution. We investigate changes in F(ab) structure by predicting theoretical scattering profiles for atomistic structures extracted from molecular dynamics (MD) simulations of the F(ab) and assessing the agreement of these structures to our experimental SAXS data. Through principal component analysis, we are able to extract principal motions observed during the MD trajectory and evaluate the influence of these motions on the agreement of structures to the F(ab) SAXS data. Changes in the F(ab) elbow angle were found to be important to reach agreement with the experimental data; however, further discrepancies were apparent between our F(ab) structure from the crystal complex and SAXS data. By analyzing multiple MD structures observed in similar regions of the principal component analysis, we were able to pinpoint these discrepancies to a specific loop region in the F(ab) heavy chain. This method, therefore, not only allows determination of global changes but also allows identification of localized motions important for determining the agreement between atomistic structures and SAXS data. In this particular case, the findings allowed us to discount the hypothesis that structural changes were induced upon complex formation, a significant find informing the drug development process. The methodology described here is generally applicable to deconvolute global and local changes of macromolecular structures and is well suited to other systems.

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Figures

Figure 1
Figure 1
Comparison of the 6G08 F(ab) from the 6G08:CD32b crystal complex to SAXS data for the 6G08 F(ab) alone. (a) The crystal structure of the 6G08 F(ab) (green) in complex with CD32b (gray) is shown. Elbow angle between constant and variable regions is measured between the twofold pseudo symmetry axes of each domain (black), with domains split at the residues denoted by the red and green balls for the heavy and light chains, respectively. The two views are rotated around a vertical axis in the paper plane. (b) The experimental scattering intensity profile of 6G08 F(ab) (gray) is overlaid with the theoretical scattering profile calculated for the 6G08 F(ab) structure from the crystal complex (green). (c) The 6G08 F(ab) crystal structure is compared to a truncated SAXS data set with a maximal q value of 0.2 Å−1. The bottom panels (b and c) show the residual plots for the respective fits; with the residuals defined as log I(q)exp – log I(q)mod.χ2 scores are calculated in CRYSOL. To see this figure in color, go online.
Figure 2
Figure 2
PCA of 6G08 F(ab) structures extracted from MD simulations identifies conformations with good agreement to SAXS data. (a–c) Structures extracted from MD simulations at 1 ns intervals are projected onto PC axes for the first three PCs. Color indicates χ2 fit to the truncated 6G08 F(ab) SAXS data, as detailed in the main text. (d–f) Histograms (gray) represent the total distribution of structures across the individual PC axes, and line plots of density distributions show structures in each χ2 fit category (black, green, orange, and red). Distributions are normalized to the individual population of each subset. (g–i) Motions captured by each PC; the C-terminal and N-terminal residues from the heavy and light chain, respectively, were removed for visualization. Arrows show general direction of motions and are not drawn to scale. Videos of these motions are available as Videos S1, S2, and S3. To see this figure in color, go online.
Figure 3
Figure 3
For a Figure360 author presentation of Fig. 3, see the figure legend at https://doi.org/10.1016/j.bpj.2018.03.040. Structures with similar PC values display different agreements to SAXS data depending on the conformation of a flexible loop in the F(ab) heavy chain. (a) All representative structures from hierarchical clustering of the MD trajectories are projected onto the original PC1 vs. PC2 axes. Colors indicate agreement of the representative structure to the truncated 6G08 F(ab) SAXS data, as previously described. Black box highlights clusters 7 (green) and 13 (black), which neighbor each other and are further analyzed in this figure. (b) Overlay of representative structures are shown for clusters 7 (green highlight) and 13 (black highlight). The major difference between structures is the position of a flexible loop, as shown in the inset. (c and d) Theoretical SAXS scattering profile of the representative frame from cluster 13 or cluster 7, respectively, compared to the truncated 6G08 F(ab) SAXS data. Bottom panels show the residual plots for the respective fits, with residuals defined as log I(q)exp – log I(q)mod. χ2 scores are calculated in CRYSOL. (e) Difference matrices show the difference between residue contributions to scattering intensity between clusters 13 and 7 at a q value of 0.1 Å−1. Colored pixels represent a difference in residue contributions and are observed most clearly at residues 136–145, corresponding to the flexible loop identified in (b).To see this figure in color, go online. .
Figure 4
Figure 4
Cluster 6 shows best agreement with the 6G08 F(ab) SAXS data. (a) Comparison of cluster 6 (black) to the truncated SAXS data for the 6G08 F(ab) (gray) is shown. The model shows good agreement to the data with a χ2 score of 1.17. Bottom panel shows the residual plots for the respective fit; residuals are defined as log I(q)exp – log I(q)mod.χ2 scores are calculated in CRYSOL. (b) Structure of the representative frame for cluster 6 (frame 2065), which has an F(ab) elbow angle of 153°, is shown. Second structure represents cluster 6 rotated to better show the elbow angle between F(ab) domains. To see this figure in color, go online.
Figure 5
Figure 5
Conformation of the flexible loop at residues 136–145 in the F(ab) heavy chain is responsible for the difference between 6G08 F(ab) crystal structure and SAXS data. (a) 6G08 F(ab) crystal structure (red) is projected onto the PC1 and PC2 axes from the original PCA of frames extracted from MD simulations. A model with similar PC values shows improved agreement with the 6G08 F(ab) SAXS data (frame 2795, orange). (b) Bound form of the 6G08 F(ab) is shown isolated from the crystal complex aligned to frame 2795. The major difference between structures is the conformation of the loop between residues 136 and 145 in the F(ab) heavy chain, shown in the inset. (c) Theoretical SAXS scattering profile of frame 2795 (χ2 7.1) is shown compared to the truncated 6G08 F(ab) SAXS data. Bottom panel shows the residual plot for the respective fit; residuals are defined as log I(q)exp – log I(q)mod.χ2 scores are calculated in CRYSOL. (d) Difference matrix between the 6G08 F(ab) crystal structure and frame 2795 at a q value of 0.1 Å−1. Colored pixels represent a difference in residue contributions and are again observed at residues 136–145. To see this figure in color, go online.
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