However, that study was limited to identifying water sites based on radial and angular distribution functions around expected H-binding atoms in the protein, while our identification was unbiased by prior expectations and identified all sites with high water density regardless of their position relative to any hydrogen bonding partners in the protein.
The side chain of Asp374 does not have a hydrogen-bonding partner in the uncomplexed TmPurL, and appears to hydrogen bond to Arg189 of TmPurQ in the complex.
In fact, the interaction network around the mandelonitrile-OH suggests that His497-Nε2 His497-Nε2nated, sisce Cys328 and Tyr457 both have to act as hydrogen bond donors to the mandelonitrile-OH for lack of any other hydrogen-bonding partner in their vicinity.
Upon ligand binding, Asp90 became ordered through H-bonding with the ligand in P2, whereas its dimeric partner lacked a H-bonding partner in P2′ and remained flexible as in the unbound state.
The hydrogen bond partners in this network, which are shared by the 4HNA starting model, extend beyond the switch loops and include not only a universally conserved salt bridge between R203 (switch I) and E236 (switch II), but also several universally conserved residues in the switch II helix (E250, N255) and L7 from the upper subdomain (Y138).
To consider the directionality of the hydrogen bonds, the features were linked with their hydrogen-bond binding partners in the protein.
The simulation revealed hPR Cys891 as the sole but weak hydrogen bonding partner of progesterone in the D-ring.
Their bonding partner was identified from preliminary structures.
Nonetheless, bystander affiliation with bonding partners has been observed in other chimpanzee and bird studies (Pan troglodytes [12], [27]; Corvus frugilegus [28]).
To experimentally address the viability of the proposed hydrogen bonding partners, tyrosine 736 was mutated to phenylalanine (Y736F) and subjected to cell death assays in neuronal cells.
