Protein environment affects the water-tryptophan binding mode. MD, QM/MM, and NMR studies of engrailed homeodomain mutantsElectronic supplementary information (ESI) available: Fig. S1: regions of NOESY spectra with cross-peaks providing NOEs restraining asparagine N51 in the NMR structure; Fig. S2: typical snapshot from the restrained MD simulation of the K52E mutant of the Drosophila EnHD in the g+ conformation along the torsion angle N-CA-CB-CG of asparagine N51; Fig. S3: NOESY spectra taken a

Water molecules can interact with aromatic moieties using either their O-H bonds or their lone-pairs of electrons. In proteins, water-π interactions have been reported to occur with tryptophan and histidine residues, and dynamic exchange between O-H π hydrogen bonding and lone-pair π interactions was suggested to take place, based on ab initio calculations. Here we used classical and QM/MM molecular dynamics simulations, complemented with an NMR study, to examine a specific water-indole interaction observed in the engrailed homeodomain and in its mutants. Our simulations indicate that the binding mode between water and indole can adapt to the potential created by the surrounding amino acids (and by the residues at the DNA surface in protein-DNA complexes), and support the model of dynamic switching between the O-H π hydrogen bonding and lone-pair π binding modes. Water molecules can interact with the π-face of tryptophan either forming an O-H π hydrogen bond or by a lone-pair π interaction. Surrounding amino acids can favor the one or the other interaction type.