Abstract
Many bacteria harbor an ATP-binding cassette (ABC) transporter named EgtU specific for the human dietary antioxidant and 2-thioimidazole-containing low-molecular weight thiol ergothioneine (ET). How the solute binding domain, EgtUC, discriminates among ET and other similar molecules is unknown. Here, we use a "chimeric" mutagenesis strategy and two distantly related EgtUCs from Streptococcus pneumoniae and Helicobacter pylori to show that a suite of EgtUC alkyl CH•••S hydrogen bonds to the ET thione S atom are central determinants of molecular recognition. Small perturbations in CH•••S distance and angle give rise to sharply attenuated transport-competent ET-bound "closed" state lifetimes and increased motional disorder in the binding pocket, not around the S atom itself, but distally in weakening NH•••O hydrogen bonds. This work highlights the impact of alkyl CH•••S H bonding in a biological protein-ligand complex in water.