Extracellular loop 4 of the proline transporter PutP controls the periplasmic entrance to ligand binding sites.
The Na(+)/proline symporter (PutP), like several other Na(+)-coupled symporters, belongs to the so-called LeuT-fold structural family, which features ten core transmembrane domains (cTMs) connected by extra- and intracellular loops. The role of these loops has been discussed in context with the gating function in the alternating access model of secondary active transport processes. Here we report the complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that reveals the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to cTM1 close to the ligand binding sites. We propose that ligand-induced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate.
Division of Microbiology, Department Biology I, LMU Munich, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.
Michael
M
Raba
Department of Physics, University of Osnabrück, Barbarastrasse 7, 49076 Osnabrück, Germany.
Sabrina
S
Dunkel
Division of Microbiology, Department Biology I, LMU Munich, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.
Daniel
D
Hilger
Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland.
Kamila
K
Lipiszko
Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland.
Yevhen
Y
Polyhach
Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland.
Gunnar
G
Jeschke
Division of Microbiology, Department Biology I, LMU Munich, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.
Susanne
S
Bracher
Department of Physics, University of Osnabrück, Barbarastrasse 7, 49076 Osnabrück, Germany.
Johann P
JP
Klare
Center for Molecular Recognition and Department of Psychiatry, Columbia University College of Physicians and Surgeons, 630 W. 168th Street, New York, NY 10032, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032, USA.
Matthias
M
Quick
Division of Microbiology, Department Biology I, LMU Munich, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany. Electronic address: hjung@lmu.de.
Heinrich
H
Jung
Department of Physics, University of Osnabrück, Barbarastrasse 7, 49076 Osnabrück, Germany. Electronic address: hsteinho@uos.de.
Heinz-Jürgen
HJ
Steinhoff
D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D008024:Ligands; D027981:Symporters; C060609:PutP protein, E coli; D010649:Phenylalanine