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J. Biol. Chem., Vol. 281, Issue 51, 39492-39498, December 22, 2006

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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. Winkler¹, and Mike Merrick²

From the Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk, NR4 7UH, United Kingdom and the Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen, Switzerland

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.

Received for publication, August 31, 2006

The atomic coordinates and structure factors (codes 2NMR, 2NOP, 2NPC, 2NOW, 2NPD, 2NPE, 2NPG, 2NPJ, and 2NPK) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the BBSRC (to A. J. and M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.

¹ Supported by the Swiss National Science Foundation within the framework of the NCCR Structural Biology program.

² To whom correspondence should be addressed. Tel.: 44-1603-450749; Fax: 44-1603-450778; E-mail: mike.merrick{at}bbsrc.ac.uk.

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