PDBsum entry 2ja3

Transport protein

PDB id

2ja3

Contents

			Protein chain

					(+ 0 more) 167 a.a.

			Ligands

					ADP ×6

References listed in PDB file

Key reference

Title

Nucleotide recognition by the cytoplasmic domain of the human chloride transporter clc-5.

Authors

S.Meyer, S.Savaresi, I.C.Forster, R.Dutzler.

Ref.

Nat Struct Biol, 2007, 14, 60-67. [DOI no: 10.1038/nsmb1188]

PubMed id

17195847

Abstract

The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report the structures of the cytoplasmic domain of the human transporter ClC-5 in complex with ATP and ADP. The nucleotides bind to a specific site in the protein. As determined by equilibrium dialysis, the affinities for ATP, ADP and AMP are in the high micromolar range. Point mutations that interfere with nucleotide binding change the transport behavior of a ClC-5 mutant expressed in Xenopus laevis oocytes. Our results establish the structural and energetic basis for the interaction of ClC-5 with nucleotides and provide a framework for future investigations.



	Figure 1. (a) Structure-based sequence alignment of the cytoplasmic domains of the Cl^- channels ClC-1 and ClC-0 and the Cl^- transporter ClC-5. Identical residues are highlighted in green, similar residues in yellow, residues involved in ATP binding in violet and the recognition sequence for ubiquitin ligase (ClC-5) in red. Secondary structure and numbering (ClC-5) are indicated above and below the sequences, respectively. The R-helix with the Cl^--coordinating tyrosine residue (#) preceding the domains is included in the alignment. The linker sequence between the two CBS domains and the C terminus in ClC-0 and ClC-1 have been omitted (XXX). The first residue of the crystallized construct is highlighted (^*). h, H. sapiens; t, T. marmorata; hClC-5, GenBank 116734718; tClC-0, GenBank X56758; hClC-1, GenBank M97820. (b) Ribbon representation of the ClC-0 domain. The two CBS subdomains are colored in green and blue, respectively; residues of the ubiquitin ligase recognition sequence are colored in red. The bound ATP molecule is shown as CPK model. (c) Relative arrangement of CBS domains in ClC-5 (yellow) and ClC-0 (red). For the ClC-0 arrangement, the two CBS subdomains of ClC-5 were superimposed on their respective counterparts in ClC-0. (d) Dimeric organization of two cytoplasmic domains of ClC-5 (colored as in a), as observed in the crystal structure. The ATP molecule is shown as CPK model. Two-fold axis of symmetry is indicated. All structure images were prepared with DINO (http://www.dino3d.org).		Figure 5. (a) Model of the cytoplasmic domains in a hypothetical dimeric arrangement, with the transmembrane domain viewed from the intracellular side. Gray ribbon, structure of E. coli ClC dimer (gray ribbon), which serves as a model for the transmembrane domains; green ribbon, R-helix; green spheres, bound ions; blue and red ribbons, the two domains, in arrangement observed in a homologous bacterial protein. ATP molecules are shown as CPK models. (b) Alternative model, with domain dimers in the conformation observed in the ClC-5 domain crystal form. View is from within the membrane; coloring scheme is similar to a. (c) Schematic model of a possible conformational change in ClC-5 induced by ATP binding. Left, model of the ClC-5 mutant E211A. ATP is bound to the cytoplasmic domain, stabilizing a conformation that allows Cl^- ions to flow equally well in both directions. Right, model of a mutant with compromised nucleotide-binding properties. In the absence of bound nucleotides, the cytoplasmic domains induce a conformational change in the ion-binding site via a regulatory helix of the transmembrane domain (R- helix, green) that diminishes Cl^- flow from the cytoplasm. The two subunits are colored in red and blue, respectively.

PROCHECK

	Headers