PDBsum entry 2qvk

Metal binding protein

PDB id: 2qvk

Contents

Protein chain

121 a.a. *

Waters ×104

* Residue conservation analysis

PDB id:

2qvk

Name:

Metal binding protein

Title:

The second ca2+-binding domain of the na+-ca2+ exchanger is essential for regulation: crystal structures and mutational analysis

Structure:

Sodium/calcium exchanger 1. Chain: a. Synonym: cbd2. Na(+)/ca(2+)-exchange protein 1. Engineered: yes

Source:

Canis lupus familiaris. Dog. Organism_taxid: 9615. Strain: familiaris. Gene: slc8a1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.45Å R-factor: 0.163 R-free: 0.192

Authors:

V.Chaptal,G.Mercado Besserer,J.Abramson,D.Cascio

Key ref:

G.M.Besserer et al. (2007). The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis. Proc Natl Acad Sci U S A, 104, 18467-18472. PubMed id: 17962412 DOI: 10.1073/pnas.0707417104

Date:

08-Aug-07 Release date: 13-Nov-07

Protein chain

P23685  (NAC1_CANLF) -  Sodium/calcium exchanger 1 from Canis lupus familiaris

Seq: 970 a.a.

Struc: 121 a.a.*

* PDB and UniProt seqs differ at 23 residue positions (black crosses)

DOI no: 10.1073/pnas.0707417104

Proc Natl Acad Sci U S A 104:18467-18472 (2007)

PubMed id: 17962412

The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.

G.M.Besserer, M.Ottolia, D.A.Nicoll, V.Chaptal, D.Cascio, K.D.Philipson, J.Abramson.

ABSTRACT

The Na(+)-Ca(2+) exchanger plays a central role in cardiac contractility by maintaining Ca(2+) homeostasis. Two Ca(2+)-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca(2+) binding to these regulatory domains activates the transport of Ca(2+) across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca(2+) ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca(2+)-bound (1.7-A resolution) and -free (1.4-A resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca(2+) ions in primary and secondary Ca(2+) sites. In the absence of Ca(2+), Lys(585) stabilizes the structure by coordinating two acidic residues (Asp(552) and Glu(648)), one from each of the Ca(2+)-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca(2+) ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca(2+) site completely remove Ca(2+) regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na(+)-Ca(2+) exchanger.

Selected figure(s)

Figure 2.
Fig. 2. Structure of the CBD2 Ca^2+ site. (A) The Ca^2+-binding sites of CBD2. The main chain is shown as blue ribbons. The two Ca^2+ ions and seven water molecules are depicted as green and red spheres, respectively. Primary and secondary Ca are labeled Ca1 and Ca2, respectively. Coordinating residues are displayed as a stick representation and colored by atom type (carbon, yellow; oxygen, red; nitrogen, blue). Interactions of residues with the Ca^2+ ions are represented by black dashed lines. (B) Ca^2+-free structure. Contacts (salt bridges with carboxyl residues and a hydrogen bond with a water molecule) with Lys^585 are shown with dashed black lines. The main chain is shown as yellow ribbons.

Figure 4.
Fig. 4. The mutations D552V and K585E have only modest effects on the biophysical properties of the Na^+–Ca^2+ exchanger. (A) Representative Na^+–Ca^2+ exchanger outward currents recorded from patches of oocytes expressing the indicated construct. Similar to WT, high intracellular Ca^2+ increased exchange currents and diminished the extent of Na^+-dependent inactivation of D552V and K585E. (B) Dose–response curves for cytoplasmic Ca^2+ for WT and mutant exchangers. Currents were measured at the peak amplitude. Residual current recorded in the absence of Ca^2+ has been subtracted. Each point is the average of four or five experiments.

Figures were selected by an automated process.