Secondary active transporters use the energy from electrochemical ion gradients and/or substrate gradients to mediate concentrative substrate translocation across biological membranes of all organisms. With advances in membrane protein structural biology, it has become clear that a large number of secondary active symporters and exchangers, which belong to distant families without discernable sequence identity, nonetheless, share common structural features that classify them into a single structural family, known as the LeuT-fold. This fold is characterized by 10 transmembrane helices (TMs) organized into two inverted structural repeats each containing 5 TMs. In Bridge 3 we seek to understand commonalities as well as divergence in the functional mechanisms of the LeuT-fold proteins, with a focus on the general rules of protein structural dynamics that underlie function, in the context of differences in their driving mechanism and the conformational changes associated with substrate translocation. To achieve these goals we build on the synergistic approach we have established with the study of LeuT, employing iterative computational, functional and spectroscopic methods. The working hypothesis of this Bridge is that the discovery and interpretation of mechanistic differences between LeuT and other LeuT-fold transporters depends on revealing dynamic properties enabled by local structural differences. We will use a new generation of quantitative computational approaches, in parallel with binding and flux studies, and with EPR and single-molecule fluorescence studies to measure distance between pairs of probes in different conformational states as well as the dynamics of the associated movements. The work will take advantage of specific established collaborations among the team members, and with the Computational Modeling, Spectroscopy and Instrumentation, and Protein Expression Cores. ApcT, a member of the APC family that also includes product/precursor exchangers, shares the LeuT-fold but has been reported to be a H+-dependent amino acid transporter. Interestingly, ApcT has the side chain ?-amino group of Lys158 occupying what is the Na2 site in LeuT, and it has been suggested that protonation and deprotonation of this Lys, like binding and unbinding of Na2 in LeuT, drives transport. In contrast to the profound mechanistic differences between ApcT and LeuT, the Drosophila dopamine transporter (dDAT) is closely related to LeuT in overall structure and function but differentiated by the presence of large amino and carboxy termini, which have been shown to critically modulate transporter function in the eukaryotic transporters. To understand similarities and differences in functional mechanisms for these compared LeuT- fold transporters we propose the following Specific Aims: 1) To determine how substrates, H+ and Na+ coordinate dynamics and conformational changes in the transport cycle of ApcT as compared with LeuT. 2) To integrate CW and DEER measurements in exploring the role of the amino terminus in modulating the conformational dynamics of dDAT.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54GM087519-08
Application #
9351547
Study Section
Special Emphasis Panel (ZGM1)
Project Start
2010-08-10
Project End
2019-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
8
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Boulanger, Eliot; Huang, Lei; Rupakheti, Chetan et al. (2018) Optimized Lennard-Jones Parameters for Druglike Small Molecules. J Chem Theory Comput 14:3121-3131
LeVine, Michael V; Cuendet, Michel A; Razavi, Asghar M et al. (2018) Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter. Biophys J 114:10-14
Diver, Melinda M; Pedi, Leanne; Koide, Akiko et al. (2018) Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT. Nature 553:526-529
Carnevale, Lauren N; Arango, Andres S; Arnold, William R et al. (2018) Endocannabinoid Virodhamine Is an Endogenous Inhibitor of Human Cardiovascular CYP2J2 Epoxygenase. Biochemistry 57:6489-6499
Molinarolo, Steven; Lee, Sora; Leisle, Lilia et al. (2018) Cross-kingdom auxiliary subunit modulation of a voltage-gated sodium channel. J Biol Chem 293:4981-4992
Paz, Aviv; Claxton, Derek P; Kumar, Jay Prakash et al. (2018) Conformational transitions of the sodium-dependent sugar transporter, vSGLT. Proc Natl Acad Sci U S A 115:E2742-E2751
Brugarolas, Pedro; Sánchez-Rodríguez, Jorge E; Tsai, Hsiu-Ming et al. (2018) Development of a PET radioligand for potassium channels to image CNS demyelination. Sci Rep 8:607
Mahinthichaichan, Paween; Morris, Dylan M; Wang, Yi et al. (2018) Selective Permeability of Carboxysome Shell Pores to Anionic Molecules. J Phys Chem B 122:9110-9118
Li, Jing; Ostmeyer, Jared; Cuello, Luis G et al. (2018) Rapid constriction of the selectivity filter underlies C-type inactivation in the KcsA potassium channel. J Gen Physiol 150:1408-1420
Kerr, Daniel; Tietjen, Gregory T; Gong, Zhiliang et al. (2018) Sensitivity of peripheral membrane proteins to the membrane context: A case study of phosphatidylserine and the TIM proteins. Biochim Biophys Acta Biomembr 1860:2126-2133

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