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PDBsum entry 2qvk
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Metal binding protein
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PDB id
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2qvk
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References listed in PDB file
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Key reference
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Title
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The second ca2+-Binding domain of the na+ ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
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Authors
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G.M.Besserer,
M.Ottolia,
D.A.Nicoll,
V.Chaptal,
D.Cascio,
K.D.Philipson,
J.Abramson.
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Ref.
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Proc Natl Acad Sci U S A, 2007,
104,
18467-18472.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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