Effect of breaking the Asp163–Lys300 salt bridge on the pKa of conserved residues. (A) Distributions of pKa values estimated with PROPKA 3.1 (Søndergaard et al., 2011) from all MD simulations shown as violin plots. pKa values were calculated from snapshots extracted at 1-ns intervals, with sodium ions included within 6 Å of the protein. For reference, a dotted line has been drawn at pH 7.6. The percentiles of the data are shown as broken lines inside the distributions, which were computed as Gaussian kernel density estimates (see Materials and methods). Data were split depending on the state of the salt bridge (formed if the distance is <4 Å, and broken if it is ≥4 Å; the salt-bridge distance distribution is also shown in yellow). The multimodal distribution of Asp164 when the salt bridge is formed is a result of data from simulations S1 (Fig. S9 A) during which its solvent accessibility is much smaller than during other simulations (see also Fig. S3, M–O). (B) Distributions of pKa shifts (ΔpKa) when the salt bridge is broken (data aggregated over all MD simulations). The pKa of Lys300 is downshifted by −2.5 ± 1.4, and thus the charged form is destabilized. Shifts of the other residues are not significantly different from 0 (also see Table 3).
