Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jan 23;104(4):1213-8.
doi: 10.1073/pnas.0610348104. Epub 2007 Jan 12.

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel

Matthew J Conroy  1 , Anne Durand, Domenico Lupo, Xiao-Dan Li, Per A Bullough, Fritz K Winkler, Mike Merrick
Affiliations

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel

Matthew J Conroy et al. Proc Natl Acad Sci U S A. .

Abstract

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 A. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of the AmtB–GlnK complex. The surface of AmtB is shown, and GlnK is shown in cartoon representation. Each subunit of AmtB and GlnK is colored independently. (A) View from the cytoplasm along the threefold axis perpendicular to the membrane. (B) View in the membrane plane. All figures were made using PyMOL (40).
Fig. 2.
Fig. 2.
Features of cytoplasmic face of AmtB in the complex. (A) Comparison of cytoplasmic loop conformations observed in the complex with the corresponding loops in PDB entry 1U7G (5) [M3–M4 (red), M5–M6 (green), and M9–M10 (magenta) in bright and pale colors, respectively]. For clarity, the end points of the loops shown as Cα traces have been centered on the helical cylinders. CTR is shown in blue (not ordered in PDB entry 1U7G). (B) Interaction of one CTR with the cytoplasmic loops of two adjacent AmtB subunits, colored pink and blue with the CTR of the pink subunit shown in yellow. Residues involved in hydrogen bonding interactions are shown as sticks. The arrow points at the position of G393.
Fig. 3.
Fig. 3.
Features of GlnK in the complex. (A) The GlnK trimer showing the T-loops extending from the main body of the protein. R47 and Y51 are shown as sticks. One molecule of ADP in the nearest lateral cleft (between the green and red subunits) is shown in stick representation. Residues at the base of the T-loop and of the B-loop from the red subunit together with residues of the C-loop from the green subunit contribute to this binding site. (B) Comparison of T-loop conformations observed in the complex (green) with the corresponding loops in E. coli GlnK (PDB entry 1GNK) (21) (pink) and E. coli GlnB (PDB entry 2PII) (26) (yellow). R47 and Y51 are indicated. Superimpositions were carried out by using backbone atoms of residues 1–35 and 58–103.
Fig. 4.
Fig. 4.
Stereo representation of the ADP binding site of GlnK. The difference electron density, contoured at 3σ, was obtained after 20 cycles of restrained refinement with the ADP molecule omitted. Residues of the two GlnK subunits forming the binding site and ADP are shown in gray, cyan, and magenta ball-and-stick models, respectively. Hydrogen bonding interactions including one water molecule (pink sphere) are shown with dashed lines, and selected residues are labeled.
Fig. 5.
Fig. 5.
Interactions of the GlnK T-loop with AmtB and consequent pore blockage. (A) Two AmtB subunits are shown in surface representation, and two GlnK molecules with ADP at their interface are in cartoon representation. The green T-loop (arrow marks kink at R38) inserts into the vestibule of the pink AmtB subunit. The surfaces of its cytoplasmic loops interacting with the T-loop are emphasized in red (M7–M8) and salmon (M5–M6), respectively, and that of the C-terminal residues of the neighboring subunit are emphasized in dark blue. (B) Hydrogen bonding interactions of the T-loop (magenta) inserted into an AmtB subunit (blue) with part of the cytoplasmic vestibule being formed by the neighboring CTR (green). Interacting residues are shown as sticks. Helices are labeled in the gray circles. H318 in the channel is included for orientation. (C) Insertion of R47 into the cytoplasmic end of the ammonia translocating channel. The channel is shown in cross-section with R47 of GlnK shown in spacefill. The rest of the T-loop is shown as a cartoon (red). H168 and H318 in the channel are shown for orientation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. von Wirén N, Merrick M. Trends Curr Genet. 2004;9:95–120.
    1. Conroy MJ, Jamieson SJ, Blakey D, Kaufmann T, Engel A, Fotiadis D, Merrick M, Bullough PA. EMBO Rep. 2004;5:1153–1158. - PMC - PubMed
    1. Blakey D, Leech A, Thomas GH, Coutts G, Findlay K, Merrick M. Biochem J. 2002;364:527–535. - PMC - PubMed
    1. Thomas GH, Mullins JG, Merrick M. Mol Microbiol. 2000;37:331–344. - PubMed
    1. Khademi S, O'Connell J, III, Remis J, Robles-Colmenares Y, Miercke LJ, Stroud RM. Science. 2004;305:1587–1594. - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources