Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum.
The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry. Electron cryo-microscopy yielded a projection map at 7.5A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.
Ziegler
C
C
Department of Structural Biology, Max-Planck-Institut für Biophysik, 60528 Frankfurt am Main, Germany. christine.ziegler@mpibp-frankfurt.mpg.de
Morbach
S
S
Schiller
D
D
Krämer
R
R
Tziatzios
C
C
Schubert
D
D
Kühlbrandt
W
W
D000345:Affinity Labels; D003001:Cloning, Molecular / Q000379:methods; D003352:Corynebacterium / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D003460:Crystallization; D008081:Liposomes; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009995:Osmosis; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D027981:Symporters / Q000737:chemistry*
D000345:Affinity Labels; D008081:Liposomes; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters