*4.3. MD Simulations*

From the docking results, several probable structures of the actin–domain V complex were selected and submitted to extensive MD simulations performed with the GROMACS software (versions 5.0.2 and 2016.1) [39]. Each selected complex initial conformation was put and solvated in a dodecahedral rhombic box of 14.0 nm edge, then neutralized by adding 175 sodium and 176 chloride ions to reach the salt concentration of 150 mM. The non-bonded interactions were treated using the smooth PME method [40] for the electrostatic terms and a cutoff distance of 1.2 nm for the van der Waals potentials. The solute and water covalent bond lengths were kept constant using the LINCS [41] and SETTLE [42] algorithms, respectively, allowing to integrate the equations of motion with a 2 fs time step. All simulations were run in the NPT ensemble, at T = 310 K and P = 1 bar, using the Nose–Hoover and Parrinello–Rahman algorithms [43–45] with the time coupling constants *τ<sup>T</sup>* = 0.5 ps and *τ<sup>P</sup>* = 2.5 ps.

In our previous study of the free state N-WASP domain V, short preliminary MD simulations indicated that the force field AMBER-03w [46] combined with the modified water model TIP4P/2005s [47] (a combination referred to as A03ws) allowed correctly exploring the protein conformational space. For consistency, we kept this force field for the study of its complex with actin. Each selected complex was submitted to about 350 ns MD simulations within the general conditions previously described. Data collected every 20 ps were kept for subsequent analyses. The latter were made using mostly the GROMACS tools, such as *gmx mindist* or *gmx cluster* for computing specific distances or structural clusters, respectively. The program STRIDE [48] was used to assign secondary structures to the protein residues.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/20/18/4493/ s1. Table S1: List of proteins with WH2 motifs which were co-crystallized with actin; Figure S1: Alignment of the studied construct sequence with those of N-WASP in 2VCP and 3M3N structures; Figure S2: N-WASP domain V residue probabilities to be in *α*-helix and distributions of gyration of the 7030 conformations in the most probable 1:1 actin–domain V complexes; Table S2: Most probable 1:1 actin–domain V encounter complexes in which domain V segment 9–18 is in contact with at least six over nine actin hot-spot residues; Table S3: Most probable 1:1 actin–domain V encounter complexes in which domain V segment 37–46 is in contact with at least six over nine actin hot-spot residues; Figure S3: N-WASP domain V residue probabilities to be in *α*-helix and distributions of gyration of the 7540 conformations in the most probable 2:1 actin–domain V complexes; Table S4: Most probable 2:1 actin–domain V encounter complexes in which segment 9–18 is in contact with at least six over eight actin hot-spot residues; Table S5: Most probable 2:1 actin–domain V encounter complexes in which segment 37–46 is in contact with at least six over eight actin hot-spot residues; Figure S4: 2/3B*best* score of N-WASP segment 433–451 redocked into actin as a function of the ligand RMSD relative to the conformation found in structure 2VCP.

**Author Contributions:** Conceptualization, M.C.-Y.-C., D.D. and T.H.-D.; Formal analysis, M.C.-Y.-C. and T.H.-D.; Funding acquisition, D.D. and T.H.-D.; Investigation, M.C.-Y.-C. and T.H.-D.; Methodology, M.C.-Y.-C., D.D. and T.H.-D.; Project administration, D.D. and T.H.-D.; Resources, D.D. and T.H.-D.; Supervision, D.D. and T.H.-D.; Validation, M.C.-Y.-C. and T.H.-D.; Visualization, M.C.-Y.-C., D.D. and T.H.-D.; Writing—original draft, M.C.-Y.-C., D.D. and T.H.-D.; and Writing—review and editing, M.C.-Y.-C., D.D. and T.H.-D.

**Funding:** This research supported by the "IDI 2016" project funded by the IDEX Paris-Saclay (grant number ANR-11-IDEX-0003-02). MD simulations were performed using HPC resources from GENCI-CINES (grant number A0040710415).

**Acknowledgments:** We are grateful to L. Renault for fruitful discussions about actin and WH2 motifs.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

*Int. J. Mol. Sci.* **2019**, *20*, 4493
