PDBsum entry 2nop

Transport protein

PDB id: 2nop

Contents

Protein chain

363 a.a. *

Ligands

SO4

ACT

IMD

GOL

Waters ×75

* Residue conservation analysis

PDB id:

2nop

Name:

Transport protein

Title:

An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance

Structure:

Ammonia channel. Chain: a. Synonym: ammonia transporter. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: amtb. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PDB file)

Resolution:

2.00Å R-factor: 0.161 R-free: 0.178

Authors:

D.Lupo,F.K.Winkler

Key ref:

A.Javelle et al. (2006). An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance. J Biol Chem, 281, 39492-39498. PubMed id: 17040913 DOI: 10.1074/jbc.M608325200

Date:

26-Oct-06 Release date: 14-Nov-06

Protein chain

P69681  (AMTB_ECOLI) -  Ammonium transporter AmtB from Escherichia coli (strain K12)

Seq: 428 a.a.

Struc: 363 a.a.

DOI no: 10.1074/jbc.M608325200

J Biol Chem 281:39492-39498 (2006)

PubMed id: 17040913

An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance.

A.Javelle, D.Lupo, L.Zheng, X.D.Li, F.K.Winkler, M.Merrick.

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.

Selected figure(s)

Figure 2.
FIGURE 2. AmtB variant expression in vivo. Extracts from GT1000 ( glnK amtB) expressing plasmid-encoded AmtB were used. A, Western blots using an anti-AmtB antibody, after SDS-PAGE of whole-cell extracts (WC), cytoplasmic (C), and membrane (M) fractions. WT, wild type. B, as in A but using anti-GS antibody.

Figure 6.
FIGURE 6. Residual difference electron densities (contoured at 3 ) in the pore region. Weighted difference density maps using (mFobs-DFcalc) terms as defined in Refmac (25) were used. Only the density peaks within a generous box (7 x 10 x 22 Å) covering the central pore region are displayed. Selected side chains at the periplasmic pore entry and along the pore are shown as ball-and-stick models. Positions corresponding to sites Am1-Am4 (7) are shown in black (S1-S4). A, difference densities from two native crystals in blue and red (buffer containing imidazole). B, difference densities and the side chains of residues 168 and 318 for the H168F and H318F in blue and red, respectively. C, difference densities and the refined side chains of residues 168 and 318 for H168A and H168E in blue and red, respectively. Except for H168A, all mutant crystals contain imidazole. The S4 peak position observed here is always about 1 Å closer to the cytoplasmic side than the Am4 site.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 39492-39498) copyright 2006.

Figures were selected by an automated process.