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.
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Research Article|
May 31 2013
Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding
Radika Soysa;
Radika Soysa
*Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, U.S.A.
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Hanka Venselaar;
Hanka Venselaar
†Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, UMC Street Radbout Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands
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Jacqueline Poston;
Jacqueline Poston
*Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, U.S.A.
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Buddy Ullman;
Buddy Ullman
*Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, U.S.A.
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Marie-Pierre Hasne
Marie-Pierre Hasne
1
*Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, U.S.A.
1To whom correspondence should be addressed (email hasnem@ohsu.edu).
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Publisher: Portland Press Ltd
Received:
March 04 2013
Revision Received:
March 21 2013
Accepted:
March 28 2013
Accepted Manuscript online:
March 28 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 452 (3): 423–432.
Article history
Received:
March 04 2013
Revision Received:
March 21 2013
Accepted:
March 28 2013
Accepted Manuscript online:
March 28 2013
Citation
Radika Soysa, Hanka Venselaar, Jacqueline Poston, Buddy Ullman, Marie-Pierre Hasne; Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding. Biochem J 15 June 2013; 452 (3): 423–432. doi: https://doi.org/10.1042/BJ20130350
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