Genetic Evidence for an Essential Oscillation of Transmembrane-Spanning Segment 5 in the Escherichia coli Ammonium Channel AmtB

Abstract

Ammonium channels, called Amt or Mep, concentrate NH 1 4 against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus ($1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH 3 to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.

The pivotal twin histidines and aromatic triad of the Escherichia coli ammonium channel AmtB can be replaced

“…The W148 variants were exceptions. Previous work indicated that W148 restricts substrate flux, and as is shown here, replacing this residue with L increases CH 3 NH 3 + conductance (2,20). These studies also provided evidence that the W148L substitution improves the rate of NH 4 + transport.…”

“…As has been observed previously (19,20), and for reasons yet to be explored, most active AmtB variants reached a slightly higher final OD 420 than the wild-type strain when grown on low-NH 3 medium.…”

“…Other instances of this behavior found in the literature include the C-terminal delete mutant of AmtB, which is a protein that lacks the last 24 residues of AmtB (19,22), and the Q57H derivative of Arabidopsis thaliana AMT1;1. The first of these mutant proteins transports CH 3 NH 3 + at 25-35% of the wild-type rate while allowing E. coli to grow on low NH 3 with a doubling time (∼100 min) longer than all functional mutants described here (19,20), and the second increases NH 4 + conductance but has a reduced CH 3 NH 3 + uptake activity (23). We show here that the correlation between Amt proteinmediated CH 3 NH 3 + and NH 4 + transport can be completely abolished.…”

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…Based on genetic evidence, an interesting idea was put forward that the C-terminal tail of AmtB facilitates an oscillation of transmembrane segment 5 (TM5) that controls the opening of both the periplasmic gate and the cytoplasmic exit (302). It is tempting to speculate that the synchronized flipping of the Phe gate (285) is related to TM5 oscillation.…”

“…The structure of the CTR was not resolved in the X-ray structures of E. coli AmtB (271,275). Deletion of the entire region substantially reduces AmtB activity to an intermediate (25 to 30%) level (269,302,303) and prevents association with GlnK (303). The addition of PhoA or LacZ peptides to the C terminus completely abolishes methylamine (MA) transport activity (270).…”