Substrate recognition in the Escherichia coli ammonia channel AmtB: a QM/MM investigation

Although the Escherichia coli ammonia transporter B (AmtB) protein has been the focus of several recent studies, there are still many questions and controversies regarding substrate binding and recognition. Specifically, how and where AmtB differentiates between substrates is not yet fully understood. The present computational study addresses the importance of intermolecular interactions with respect to substrate recruitment and recognition by means of ab initio QM/MM simulations. On the basis of calculations with substrates NH(3), NH(4)(+), Na(+), and K(+) positioned at the periplasmic binding site (Am1) and NH(3) and NH(4)(+) at intraluminal binding sites (Am1a/b), we conclude that D160 is the single most important residue for substrate recruitment, whereas cation-π interactions to W148 and F107 are found to be less important. Regarding substrate recruitment and recognition, we find that only NH(4)(+) and K(+) reach the Am1 site. However, NH(4)(+) has the largest affinity for this site due to its better dehydration compensation, while charge stabilization effects favor the binding of NH(4)(+) over NH(3) (i.e., if NH(3) would enter the Am1 site, it is likely to be protonated). Therefore, we conclude that the Am1 site selects NH(4)(+) over Na(+), K(+) and NH(3). Our calculations also suggest that translocation of NH(4)(+) from Am1 into the channel lumen is driven by rotation of the A162-G163 peptide bond, which coordinates NH(4)(+) but not NH(3) at both Am1 and Am1a/b sites.