Dissertations, Theses, and Capstone Projects

Date of Degree

2-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Marilyn R. Gunner

Committee Members

Alessio Accardi

Ranajeet Ghose

Themis Lazaridis

Shaneen Singh

Subject Categories

Biochemistry | Bioinformatics | Biophysics

Keywords

CLC exchangers, proton-coupled Cl¯ binding, titration simulation, MCCE

Abstract

The CLC family of membrane proteins is a ubiquitously expressed class of proton and usually voltage-activated chloride transporters involved in a myriad of physiological functions. Crystallographic structures identify up to three chloride binding sites: external, central and intracellular located in the inner half of the trans-membrane domain. The CLC proteins, except for the kidney isoforms, are gated by the extracellular side-facing gating Glutamate (Ex, E148 in CLC-ec1, the E. coli exchanger), which is thought to undergo a conformational change upon protonation.

To sort out how the thermodynamic paths to H+ coupled Cl¯ binding and conformational change in CLC-ec1 at the extracellular site are related, we constructed possible transport cycle intermediates with varied Ex conformations and used Multi-Conformational Continuum Electrostatics (MCCE) to simulate ligand titrations. We find that Cl¯ binding is strongly coupled to H+ binding at the external site; the Cl¯ affinity is linearly correlated with the proton affinity, whose range is about 11 kcal/mol while the range for the Cl¯ affinity is about 20 kcal/mol, ensuring Cl¯ binding and release with minimal protein rearrangement.

The wide-ranging apo proton-affinity of Ex is almost entirely controlled by its interaction energy with the protein backbone and is conformation-dependent: the outward open conformation is where the backbone interaction energy with Ex is minimal and proton affinity is maximal. The 'helix cage' topology of the two sites in the selectivity filter, characterized by the electropositive hotspot resulting from the abutting of the N-termini of helix F and N, enables the binding of two negative charges and explains how two Cl ions can be bound within ~ 4 Å of each other, in spite of strong ion-ion repulsion. Chloride binding data for the central site show that the central site affinity is not correlated to the Ex apo pKa. We find that this site has a lower affinity than the exterior site. While Cl¯ binding at the central site is also stabilized by backbone elements, it is hindered by rearrangements of S107 and Y445 that are often necessary upon Cl binding.

The main significance of this study of H+ and Cl¯ binding in occluded structures resides in showing that binding at the external site initiates the H+/Cl¯ coupling and that the higher proton affinity of the gating residue once it leaves the helix cage enables two Cl ions to bind in close proximity. Thus this study provides atomistic details of the experimentally measured 2Cl¯:1H+ stoichiometry of binding in CLC-ec1.

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