Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction.

Jinan Wang,Qiang Shao,Tim Fulford,Huaiyu Yang,Weiliang Zhu,Mike Merrick,Arnaud Javelle

doi:10.1371/journal.pone.0062745

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.

Highlights

[8] This phenotype can be complemented by heterologous expression of ammonium transport (Amt) proteins from a variety of organisms. [44,47,64] So we expressed wild-type Escherichia coli AmtB (EcAmtB) and the variants H168A, H318A, H168A/H318A in S. cerevisiae strain 31019b
We confirmed that all the EcAmtB variant proteins were present in the S. cerevisiae cell membrane by Western blotting of membrane fractions using an anti-EcAmtB antibody
The necessity for the presence of an aromatic ring at residue 212 was demonstrated in our earlier studies, where we showed that a W212F variant of EcAmtB is active but a W212A variant is not. [23,33] Once at site Am4, ammonia could exit from H168A EcAmtB

Summary

Introduction

Ammonium transport is facilitated by a highly conserved family of membrane proteins, represented by the ammonium transport (Amt) proteins in bacteria,[1,2,3,4] plants,[5,6,7] and yeast (where they are designated methylamine permease or Mep proteins), [8,9] and by the Rhesus (Rh) proteins in animals. [10,11] High resolution structures have been determined for Escherichia coli AmtB (EcAmtB), [12,13] Amt-1 from Archaeoglobus fulgidus, [14] Rh50 from Nitrosomonas europaea, [15,16] and human RhCG, [17] all of which show considerable structural conservation. [30,31] Computational simulations have focussed on EcAmtB and have predominantly supported the electroneutral NH3 transport model These simulations suggested that prior to electroneutral ammonia being transported into the cytoplasm, an NH4+ ion is bound in the extracytoplasmic vestibule and is subsequently deprotonated by a mechanism that is still a matter of debate.[25,32,33,34,35,36,37,38,39,40,41,42,43]. The pore of EcAmtB is lined with hydrophobic residues, and there is a pair of histidines, His168 and His318, near the mid-point These two histidines are highly conserved in both the Amt and Rh families, and they have been postulated to play a critical role in mediating ammonia transport

Methods

Results

Conclusion