Structure of sugar-bound LacY.
Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds D-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.
Hemant
H
Kumar
Vladimir
V
Kasho
Irina
I
Smirnova
Janet S
JS
Finer-Moore
H Ronald
HR
Kaback
Robert M
RM
Stroud
D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D007544:Isopropyl Thiogalactoside / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013379:Substrate Specificity
D000596:Amino Acids; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D007544:Isopropyl Thiogalactoside; C021499:lactose permease
X-ray structure; conformational change; induced fit; membrane protein; transport