An Unusual Twin-His Arrangement in the Pore of Ammonia Channels Is Essential for Substrate Conductance

Arnaud Javelle,Domenico Lupo,Lei Zheng,Xiao-Dan Li,Fritz K Winkler,Mike Merrick

doi:10.1074/jbc.m608325200

Arnaud Javelle, Domenico Lupo + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m608325200

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Abstract

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.

Highlights

Ammonium is the preferred nitrogen source for many organisms, and its transport across cellular membranes is a fundamental biological process. (In this report, we use the term ammonium to refer to both the protonated (NH4ϩ) and the unprotonated (NH3) forms and ammonia to refer to NH3.) Ammonium transport is mediated by a family of ubiquitous membrane proteins, found in bacteria, archaea, fungi, and plants, whose homologues in animals are the Rhesus (Rh)3 proteins [1, 2]
By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance
Position of two conserved histidine side chains in the pore and suggested that they play a critical role in substrate conductance

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis primers are listed in Supplemental Table SI. PAJ2011 and pAJ2012 were constructed using the QuikChangeTM mutagenesis kit (Stratagene). Fractionation, AmtB Purification, and Western Blotting— E. coli AmtB was purified as described [5]. Cell fractionation and Western blotting were as described [20] using polyclonal. Protein Quantification—For each AmtB variant, serial dilutions of whole-cell extracts of strain GT1000 (⌬glnK⌬amtB) expressing a mutant amtB allele were subject to Western blotting using antiAmtB antibodies. [14C]Methylammonium Transport Assays—Methylamine accumulation (AmtB activity) and methylglutamine accumulation (glutamine synthetase activity) were determined as described [10]

This work

Data collection

RESULTS

DISCUSSION