Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data

Abstract

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.

Figure 1

Sequence alignment of N-WASP domain VC crystallized with actin by Gaucher et al. (2VCP_D) with the 67-residue peptide used in this study (domain V). The upper amino acid numbering comes from the human sequence. The solid line box indicates the 19 residues of the second WH2 motif present in the x-ray structure (34). The dashed line box highlights the homologous 19 residues of the first WH2 motif. To see this figure in color, go online.

Figure 2

Comparison between SAXS intensity back-calculation software using implicit-solvent models versus WAXSiS, which uses an explicit-solvent approach. Orange, green, blue, and pink bars are results for CRYSOL, FoXS, CRYSOL3, and Pepsi-SAXS, respectively. Shaded bars indicate results when using default values for the hydration layer density. To see this figure in color, go online.

Figure 3

Time evolution of N-WASP domain V radius of gyration computed from short MD simulations using six different force fields. Horizontal colored dashed lines and black solid lines indicate average radii of gyration in simulations and in experiments, respectively. To see this figure in color, go online.

Figure 4

Direct comparison of SSP scores (A and B) and N-H RDCs (C and D) averaged over FM_nossp, FM_ssp, MD_C36mm, and MD_A03ws ensembles with NMR measurements. Probabilities for N-WASP domain V residues to be in α-helix (E and F) and β-strand (G and H) structures computed from the FM_nossp, FM_ssp, MD_C36mm, and MD_A03ws ensembles are shown. Dashed and dotted brown vertical lines represent protein regions in α-helix (as indicated by the x-ray structure 2VCP (34)) and the highly conserved sequences LK[K-S]V (35, 36), respectively. To see this figure in color, go online.

Figure 5

Comparison of SAXS intensities Log I(q) (A and B) and Kratky plots (C and D) as a function of scattering vector q calculated from the FM_nossp, FM_ssp, MD_C36mm, and MD_A03ws ensembles against experimental data. Reduced residuals Δ/σ = [I_calc(q) − I_exp(q)]/σ_exp(q). Probability of the N-WASP domain V radius of gyration (E and F) computed for the F_nossp, FM_ssp, MD_C36mm, and MD_A03ws conformational ensembles is shown. Vertical black solid and colored dashed lines indicate radius of gyration measured by experiments and mean values found in simulations, respectively. To see this figure in color, go online.

Figure 6

Comparison with NMR experiments of SSP scores (A and B) and N-H RDCs (C and D) averaged over FM_ssp and MD_A03ws ensembles, before and after selection by GAJOE. Residue-specific probability to be in α-helix (E and F) or in β-strand (G and H) calculated from the FM_ssp and MD_A03ws ensembles, before and after selection by GAJOE, is shown. To see this figure in color, go online.

Figure 7

Comparison with SAXS experiments of Log I(q) (A and B) and Kratky plots (C and D) averaged over the FM_ssp and MD_A03ws ensembles, before and after selection with GAJOE. Reduced residuals Δ/σ = [I_calc(q) − I_exp(q)]/σ_exp(q). Probability of the N-WASP domain V radius of gyration (E and F) computed for the FM_ssp and MD_A03ws conformational ensembles, before and after selection by GAJOE, is shown. To see this figure in color, go online.