PDBsum entry 3c1h

Transport protein

PDB id: 3c1h

Contents

Protein chain

363 a.a. *

Ligands

ACT

LDA

IMD

Waters ×130

* Residue conservation analysis

PDB id:

3c1h

Name:

Transport protein

Title:

Substrate binding, deprotonation and selectivity at the periplasmic entrance of the e. Coli ammonia channel amtb

Structure:

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

Source:

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

Resolution:

2.20Å R-factor: 0.206 R-free: 0.231

Authors:

D.Lupo,F.K.Winkler

Key ref:

A.Javelle et al. (2008). Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB. Proc Natl Acad Sci U S A, 105, 5040-5045. PubMed id: 18362341 DOI: 10.1073/pnas.0711742105

Date:

23-Jan-08 Release date: 18-Mar-08

Protein chain

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

Seq: 428 a.a.

Struc: 363 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1073/pnas.0711742105

Proc Natl Acad Sci U S A 105:5040-5045 (2008)

PubMed id: 18362341

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

A.Javelle, D.Lupo, P.Ripoche, T.Fulford, M.Merrick, F.K.Winkler.

ABSTRACT

The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH(4)(+) deprotonation takes place at S1 before NH(3) conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

Selected figure(s)

Figure 4.
Periplasmic pore constriction of AmtB wild type and variants. The solvent-accessible space shown as a light blue surface was calculated by using the program CAVER, with a water-omitted structure. (A) F107A. (B) F215A. (C) F107A/F215A. Selected, highly conserved residues are shown in ball-and-stick representation for the ammonium binding site, phenylalanine gate, and central pore. The substitutions are labeled in red.

Figure 6.
Proton and potassium conductance of liposomes and proteoliposomes. Shown is pH variation of liposomes (blue trace) and proteoliposomes containing wild-type AmtB (black trace) or the double variant F107A/F215A (red trace) when applying a pH pulse of 1 unit by adding 5 mM KOH. The dashed line indicates addition of 1 μM valinomycin in A and 1 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone) in B. The arrow on each trace indicates the addition of 1 μM FCCP in A and 1 μM valinomycin in B. The alkalanization rate constant (k, s^−1) resulting from the charge equilibration due to the equilibration of H^+ (A) and K^+ movement (B) is noted above each trace. The apparent proton permeabilities P′ [H], measured as described in the text, were 2.0 × 10^−8, 2.8 × 10^−8, and 2.0 × 10^−8 cm/s for liposomes and proteoliposomes containing wild-type AmtB or the double variant, respectively. The apparent K^+ permeabilities P′ [K] were 1.5 × 10^−8, 2.3 × 10^−8, and 2.5 × 10^−8 cm/s. Experiments were performed at 20°C.

Figures were selected by an automated process.