title: Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction.
abstract: Two conserved histidine residues are located near the mid-point of the conduction channel of ammonium transport proteins. The role of these histidines in ammonia and methylamine transport was evaluated by using a combination of in vivo studies, molecular dynamics (MD) simulation, and potential of mean force (PMF) calculations. Our in vivo results showed that a single change of either of the conserved histidines to alanine leads to the failure to transport methylamine but still facilitates good growth on ammonia, whereas double histidine variants completely lose their ability to transport both methylamine and ammonia. Molecular dynamics simulations indicated the molecular basis of the in vivo observations. They clearly showed that a single histidine variant (H168A or H318A) of AmtB confines the rather hydrophobic methylamine more strongly than ammonia around the mutated sites, resulting in dysfunction in conducting the former but not the latter molecule. PMF calculations further revealed that the single histidine variants form a potential energy well of up to 6 kcal/mol for methylamine, impairing conduction of this substrate. Unlike the single histidine variants, the double histidine variant, H168A/H318A, of AmtB was found to lose its unidirectional property of transporting both ammonia and methylamine. This could be attributed to a greatly increased frequency of opening of the entrance gate formed by F215 and F107, in this variant compared to wild-type, with a resultant lowering of the energy barrier for substrate to return to the periplasm.
authors.affiliation: Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jinan
authors.initials: J
authors.lastname: Wang
authors.forename: Tim
authors.initials: T
authors.lastname: Fulford
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shao
authors.forename: Arnaud
authors.initials: A
authors.lastname: Javelle
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Yang
authors.forename: Weiliang
authors.initials: W
authors.lastname: Zhu
authors.forename: Mike
authors.initials: M
authors.lastname: Merrick
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008744:Methylamines / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C107270:MEP2 protein, S cerevisiae; D008744:Methylamines; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D000641:Ammonia; C027451:methylamine