title: Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.
abstract: The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
authors.forename: Radika
authors.initials: R
authors.lastname: Soysa
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Poston
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Hasne
mesh_terms: D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000378:metabolism; D002103:Cadaverine / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011237:Predictive Value of Tests; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011700:Putrescine / Q000737:chemistry* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D026905:Amino Acid Transport Systems; D008024:Ligands; D026901:Membrane Transport Proteins; D002103:Cadaverine; D011700:Putrescine
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