Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger

Journal Article · Sun Jan 01 00:00:00 EST 2006 · Journal of Molecular Biology

DOI:https://doi.org/10.1016/j.jmb.2006.07.081· OSTI ID:930095

Accardi, A; Lobet, S; Williams, C; Miller, C; Dutzler, R

The Cl{sup -}/H{sup +} exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl{sup -} ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl{sup -} ions visible in the structure of this protein and is located near the intersection of the Cl{sup -} and H{sup +} pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl{sup -} and H{sup +} determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl{sup -} transport but greatly impaired movement of H{sup +}+. The obligatory 2 Cl{sup -}/1 H{sup +} stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl{sup -} binding region correlates with functional H{sup +} coupling. In particular, as determined by anomalous diffraction in crystals grown in Br{sup -}, an electrophysiologically competent Cl{sup -} analogue, the well-coupled transporters show strong Br{sup -} electron density at the 'inner' and 'central' Cl{sup -} binding sites. However, in the uncoupled mutants, Br{sup -} density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H{sup +} for Cl{sup -}, but at a reduced H{sup +}-to-Cl{sup -} ratio; likewise, both the central and inner sites are occupied by Br{sup -}, but the central site shows lower Br{sup -} density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H{sup +}, a synergism that is not readily understood in terms of alternating-site antiport schemes.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 930095
• Report Number(s): BNL-80733-2008-JA; JMOBAK; TRN: US0806703
• Journal Information: Journal of Molecular Biology, Vol. 362, Issue 4; ISSN 0022-2836
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS
ANIONS
COORDINATES
CORRELATIONS
COUPLING
CRYSTALS
DENSITY
DIFFRACTION
ELECTRON DENSITY
FUNCTIONALS
HALIDES
IONS
MUTANTS
PROTEIN STRUCTURE
PROTEINS
PROTON TRANSPORT
PROTONS
STOICHIOMETRY
SYNERGISM
TRANSPORT
TYROSINE
national synchrotron light source