Apart from the electrostatic effects of the metal cation and hydrogen bonding with the water molecules coordinated to this ion, the leaving group is additionally forming a strong hydrogen bond with Tyr367, a hydrogen bond with Arg362 and another, relatively weak hydrogen bond with the amidic hydrogen of the acceptor threonine.

Furthermore, water molecules present in the active site were manually rotated to create a network of hydrogen bonds where possible.

Finally, 12 highly conserved residues interacting with the substrates were included (Table 2) as well as six water molecules that were well defined in the crystal structure (B-factors below 20).

Three of these water molecules are located close to the metal ion with one of them directly serving as a ligand and the other two forming a hydrogen bond network between the first water molecule and neighboring active site residues.

The DAB residues were observed to drag some water molecules into the hydrophobic bilayer core (Fig 3); on average less than one water molecule was present in the PMB1 free membrane, compared to an average of seven being present during the last microsecond of simulation.

Moreover, when we studied the radial distribution of solvent around DAB residues, despite being fully inserted within the non-polar acyl tail region, an average of three water molecules were still present within 0.35 nm of the DAB nitrogen atoms (Fig 3).

This is further supported by analysis of the mass density profile (Fig 3) that reveals increased water penetration, with water molecules reaching the hydrophobic core of the membrane as discussed above.

Interaction of the DAB residues with the membrane surface during aggregation caused water molecules to move towards the membrane.

Radial distribution functions (S4 Fig) showed an increased degree of solvation in the sugar group area of the membrane, such that on average 20 water molecules were present in this region after 2 pi-croseconds, compared to ~12 molecules prior to PMB1 binding.