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 Title: Molecular insights into the mechanisms of transport and energy coupling in membrane transport proteins Author(s): Jiang, Tao Director of Research: Tajkhorshid, Emad Doctoral Committee Chair(s): Tajkhorshid, Emad Doctoral Committee Member(s): Gennis, Robert; Grosman, Claudio; Jin, Hong Department / Program: School of Molecular & Cell Bio Discipline: Biophysics & Computnl Biology Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Membrane transport Antiporter Membrane exchanger Channel Molecular simulation Phospholipid scrambling Mutagenesis Principal component analysis Abstract: Membrane transport proteins are the main gatekeepers controlling the traffic of molecules in and out the cell. The mechanism by which they mediate selective and regulated transport across the membrane is of broad physiological and biophysical relevance. In this dissertation, several critical aspects of the transport process have been studied through molecular dynamics (MD) simulations, including ion binding and its coupling to chemical processes such as H+ transport, translocation of the transported substrate and cotransported ions, dynamics of the catalytic site, coordinated motions of the remote regions, as well as other molecular events facilitating the transport of the cargo. The first part of the dissertation covers topics on a Cl-/H+ transporter from the CLC superfamily, which catalyzes stoichiometrically coupled exchange of Cl- and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F-, and larger physiological anions, such as PO4^3- and SO4^2-. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter have revealed that Cl- binding to the central anion-binding site is crucial for the anion-coupled H+ transport. This study shows that Cl-, F-, NO3-, and SCN- display distinct binding coordinations at the central site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl-. Another important question concerning the functional mechanism of CLC transporters is that no large conformational change have been detected crystallographically, even though transporters usually undergo global conformational change to alternately expose substrate-binding sites to opposite sides of the membrane. The collaborative work here demonstrates the formation of a previously uncharacterized `outward-facing open' state enrich by high H+ concentration, which involves global structural changes ~20 A away from the outer gate. This long distance conformational change highlights the coupled motions as well as the relevance of global structural changes in CLC transport cycle. The second part of the dissertation focus on a phospholipid scramblase which mediates rapid transbilayer redistribution (scrambling) of phospholipids at plasma membrane. This process dissipates lipid asymmetry in response to signals for critical cellular events like apoptosis that elevate cytoplasmic Ca^2+ concentration. The work here shows that the hydrophilic aqueduct on the surface of the fungal scramblase nhTMEM16 serves as the path for lipid translocation, and that Ca$^{2+}$ binding plays a key role in determining an open conformation of the path for lipid diffusion. The fully occupied lipid track connects the inner and outer leaflets and forms a “proteolipidic” pore, which allows ion conduction through the aqueous pathway formed between the protein and lipid headgroups under transmembrane electric potentials. Supporting this mechanism, site-specific mutagenesis experiments show that nhTMEM16 ionic currents are synergistically linked to phospholipid scrambling. To further validate the idea that ions permeate through TMEM16s via the same structural pathway taken by phospholipids, two specific residues in the pore region were pinpointed, which are able to convert TMEM16A Ca^2+-activated Cl- channel (CaCC) into robust scramblase upon point mutations. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s. Issue Date: 2017-07-13 Type: Text URI: http://hdl.handle.net/2142/98275 Rights Information: Copyright 2017 Tao Jiang Date Available in IDEALS: 2017-09-29 Date Deposited: 2017-08
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