Development and application of EPR-spectroscopic methods for the analysis of structure and dynamics of integral membrane proteins using the Na+/proline transporter PutP as a model
Final Report Abstract
Secondary transporters harness the energy stored in (electro)chemical gradients of a solute or ion (e.g., proton or sodium ion) to drive transport of a second solute across cell membranes. The transporters are proposed to follow an alternating-access mechanism in which the binding sites for the ligands are alternately exposed to one or the other side of the membrane. The overall aim of this research project was a contribution to the understanding of the mechanistic details of alternative access in solute/sodium symport. For this purpose, we used the sodium/proline symporter PutP, a member of the solute/sodium symporter (SSS) family (TC 2.A.21), as a model. To reach the aim, electron paramagnetic resonance (EPR) spectroscopic techniques were advanced for applications with integral membrane protein, and applied to PutP. Methodological achievements involve the setup of an EPR spectrometer solely for accessibility measurements and automated handling of the measurement of ~300 samples as function of microwave power, and the development of tools for time resolved EPR experiments. The methods were applied for a complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that revealed the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to core transmembrane domain 1 (cTM1’) close to the ligand binding sites. Based on these results we proposed that ligandinduced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate. The model was extended by implicating interactions between Glu311 at the tip of eL4 and the peptide backbone at the end of cMD10’ in external gate closure and stabilization of an inwardopen conformation. Finally, the number of substrate binding sites in the SSS members vSGLT and PutP was evaluated. A combined computational and experimental approach identified in addition to the central substrate binding a second, more externally localized substrate binding site in both SSS transporters similar as previously proposed for the neurotransmitter/sodium symporter LeuT. Replacement of amino acids in the proposed sites resulted in the reduction of the binding stoichiometry and was accompanied by severely impaired translocation of proline. Taken together, our observations further specify the idea that common design and functional principles are maintained across different transport families with similar structure.
Publications
- (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) - (2014) Extracellular loop 4 of the proline transporter PutP controls the periplasmic entrance to ligand binding sites. Structure, 22, 769-780
Raba, M., Dunkel, S., Hilger, D., Lipiszko, K., Polyhach, Y., Jeschke, G., Bracher, S., Klare, J.P., Quick, M., Jung, H. and Steinhoff, H.-J.
(See online at https://doi.org/10.1016/j.str.2014.03.011) - (2015) Identification of a second substrate binding site in solute:sodium symporters. J. Biol. Chem. 290, 127-141
Li, Z., Lee, A.S.E., Bracher, S., Jung, H., Paz, A., Kumar, J.P., Abramson, J., Quick, M. and Shi, L.
(See online at https://doi.org/10.1074/jbc.M114.584383) - (2015) Spin Labeling Studies of Transmembrane Signaling and Transport: Applications to Phototaxis, ABC Transporters and Symporters. Methods in Enzymology
Klare, J.P., and Steinhoff, H.-J.
(See online at https://doi.org/10.1016/bs.mie.2015.05.025)
