Structural basis of substrate selectivity in the glycerol-3-phosphate: phosphate antiporter GlpT.
Major facilitators represent the largest superfamily of secondary active transporter proteins and catalyze the transport of an enormous variety of small solute molecules across biological membranes. However, individual superfamily members, although they may be architecturally similar, exhibit strict specificity toward the substrates they transport. The structural basis of this specificity is poorly understood. A member of the major facilitator superfamily is the glycerol-3-phosphate (G3P) transporter (GlpT) from the Escherichia coli inner membrane. GlpT is an antiporter that transports G3P into the cell in exchange for inorganic phosphate (P(i)). By combining large-scale molecular-dynamics simulations, mutagenesis, substrate-binding affinity, and transport activity assays on GlpT, we were able to identify key amino acid residues that confer substrate specificity upon this protein. Our studies suggest that only a few amino acid residues that line the transporter lumen act as specificity determinants. Whereas R45, K80, H165, and, to a lesser extent Y38, Y42, and Y76 contribute to recognition of both free P(i) and the phosphate moiety of G3P, the residues N162, Y266, and Y393 function in recognition of only the glycerol moiety of G3P. It is the latter interactions that give the transporter a higher affinity to G3P over P(i).
Law
Christopher J
CJ
Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York, New York, USA.
Enkavi
Giray
G
Wang
Da-Neng
DN
Tajkhorshid
Emad
E
D000595:Amino Acid Sequence; D003198:Computer Simulation; D004926:Escherichia coli; D005578:Fosfomycin / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics
C488962:GlpT protein, E coli; D026901:Membrane Transport Proteins; D005578:Fosfomycin; D010744:Phosphoric Monoester Hydrolases; C040842:glycerol-1-phosphatase