The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.
TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.
Yin
C C
CC
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Aldema-Ramos
M L
ML
Borges-Walmsley
M I
MI
Taylor
R W
RW
Walmsley
A R
AR
Levy
S B
SB
Bullough
P A
PA
D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D003460:Crystallization; D004926:Escherichia coli / Q000737:chemistry* / Q000254:growth & development; D006639:Histidine / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D020836:Protein Structure, Quaternary
D017920:Antiporters; D001426:Bacterial Proteins; C079051:tetA protein, Bacteria; D006639:Histidine