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CALX-CBD1 Ca 2+ -binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics
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ABSTRACT
The Na
+
/Ca
2+
exchanger of
Drosophila melanogaster
, CALX, is the main Ca
2+
-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na
+
/Ca
2+
exchangers have two Ca
2+
sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca
2+
-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca
2+
at physiological concentrations. CALX-CBD1 consists of a
β
-sandwich and displays four Ca
2+
binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca
2+
-binding to CALX-CBD1. We observed that this domain binds Ca
2+
in the slow exchange regime at the NMR chemical shift time scale. Ca
2+
-binding restricts the dynamics in the Ca
2+
-binding region. Experiments of
15
N CEST and
15
N R
2
dispersion allowed the determination of Ca
2+
dissociation rates (≈ 20 s
−1
). NMR titration curves of residues in the Ca
2+
-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R
2
. Hence, a novel approach to analyze NMR titration curves was proposed. Ca
2+
-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded
n
Hill
= 2.9 − 3.1, near maximum cooperativity (
n
Hill
= 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca
2+
is at least three orders of magnitude higher than that of binding a single Ca
2+
. Hence, four Ca
2+
ions bind almost simultaneously to CALX-CBD1. Cooperative Ca
2+
-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca
2+
-concentration in sensory neuronal cells.
SIGNIFICANCE
CALX-CBD1 is the Ca
2+
-sensor domain of the Na
+
/Ca
2+
exchanger of
Drosophila melanogaster
. It consists of a
β
-sandwich, and contains four Ca
2+
binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca
2+
ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca
2+
with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca
2+
.
Title: CALX-CBD1 Ca
2+
-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics
Description:
ABSTRACT
The Na
+
/Ca
2+
exchanger of
Drosophila melanogaster
, CALX, is the main Ca
2+
-extrusion mechanism in olfactory sensory neurons and photoreceptor cells.
Na
+
/Ca
2+
exchangers have two Ca
2+
sensor domains, CBD1 and CBD2.
In contrast to the mammalian homologues, CALX is inhibited by Ca
2+
-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca
2+
at physiological concentrations.
CALX-CBD1 consists of a
β
-sandwich and displays four Ca
2+
binding sites at the tip of the domain.
In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca
2+
-binding to CALX-CBD1.
We observed that this domain binds Ca
2+
in the slow exchange regime at the NMR chemical shift time scale.
Ca
2+
-binding restricts the dynamics in the Ca
2+
-binding region.
Experiments of
15
N CEST and
15
N R
2
dispersion allowed the determination of Ca
2+
dissociation rates (≈ 20 s
−1
).
NMR titration curves of residues in the Ca
2+
-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R
2
.
Hence, a novel approach to analyze NMR titration curves was proposed.
Ca
2+
-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis.
Fittings of NMR and ITC binding curves to the Hill model yielded
n
Hill
= 2.
9 − 3.
1, near maximum cooperativity (
n
Hill
= 4).
By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca
2+
is at least three orders of magnitude higher than that of binding a single Ca
2+
.
Hence, four Ca
2+
ions bind almost simultaneously to CALX-CBD1.
Cooperative Ca
2+
-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca
2+
-concentration in sensory neuronal cells.
SIGNIFICANCE
CALX-CBD1 is the Ca
2+
-sensor domain of the Na
+
/Ca
2+
exchanger of
Drosophila melanogaster
.
It consists of a
β
-sandwich, and contains four Ca
2+
binding sites at the distal loops.
In this study, we examined the cooperative binding of four Ca
2+
ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments.
NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics.
A novel approach to analyze NMR titration data in the slow exchange regime was proposed.
These results support the view that CALX-CBD1 binds four Ca
2+
with high cooperativity.
The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca
2+
.
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