Structure-function analysis of the NA+/proline transporter PutP
Final Report Abstract
The Na+/solute symporter (SSS) family encompasses proteins that transport a wide variety of solutes including sugars, amino acids, vitamins, osmolytes, and ions across the plasma membrane from bacteria to man. Crystal structures of the Vibrio parahaemolyticus Na+/galactose transporter (vSGLT) are available, but critical mechanistic details of the transport cycle remain unresolved. Here, the Na+/proline transporter PutP, a bacterial member and well-established model system of the SSS family was used to relate structural features of SSS members to function using amino acid substitution, kinetic, proteinchemical and spectroscopic analyses. It was shown that TMDs 2 and 9 of PutP line the ligand translocation pathway in PutP. Structural modeling and Cys accessibility analyses revealed that the cytoplasmic halves of both TMDs form a cavity that is open to the cytoplasm in the absence of ligands. The cavity closes upon addition of Na+ and proline. Amino acids important for Na+ and proline binding are located at the inner end of the cavity. The Na+ site is formed by TMD 2 (A53, M56) and TMD 9 (A337, S340, T341); the proline binding site is more complex and involves amino acids of TMDs 2, 4, 7, and 9. Furthermore, the role of external loop 4 connecting TMDs 8 and 9 of PutP in the alternating access mechanism was investigated. A complete Cys scanning mutagenesis of the loop in combinations with spin labeling and EPR spectroscopic analyses suggests that the loop functions as a gate and controls access of the ligand binding sites from the periplasmic side of the membrane. Besides PutP of E. coli, the putative proline transporter of the human pathogen Helicobacter pylori (HpPutP) was included in the analyses. A previous investigation revealed that the respective gene is essential for stomach colonization by H. pylori. In this project, HpPutP was demonstrated to function as a high affinity Na+/L-proline symporter similar to its E. coli ortholog. Amino acids crucial for HpPutP function were identified. A screen of prolinerelated compounds revealed two potent inhibitors of HpPutP function. HpPutP represents a potential new drug target for the treatment of H. pylori infections.
Publications
- (2009) Backbone structure of transmembrane domain IX of the Na+/proline transporter PutP of Escherichia coli. Biophys. J., 96, 217-225
Hilger, D., Polyhach, Y., Jung, H. and Jeschke, G.
- (2010) Protein chemical and EPR spectroscopic approaches to monitor membrane protein structure and dynamics. in Bacterial signaling (Krämer, R. and Jung, K., Eds.), Wiley-VCH, Weinheim, pp. 247-263
Hilger, D. and Jung, H.
- (2011) Homology model of the Na+/proline transporter PutP of Escherichia coli and its functional implications. J. Mol. Biol. 406, 59-74
Olkhova, E., Raba, M., Bracher, S., Hilger, D. and Jung, H.
- (2012) The Na+/proline transporter PutP. Front. Biosci. 17, 745-59
Jung, H., Hilger, D. and Raba, M.
(See online at https://doi.org/10.2741/3955)
