Structural Analysis of Mg2+ and Ca2+ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels

Wingard, Jennifer N.; Chan, Jimmy Yu‐Wai; Bosanac, Ivan; Haeseleer, Françoise; Palczewski, Krzysztof; Ikura, Mitsuhiko; Ames, James B.

doi:10.1074/jbc.m508541200

Cited by 80 publications

(130 citation statements)

References 64 publications

(66 reference statements)

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“…The recombinant CaBP1 protein was cloned into a pET3b vector and expressed in Escherichia coli BL21(DE3) cells as described previously. 23 Uniformly 13 C/ 15 N-labeled protein expression was induced by the addition of 0.5 mM IPTG at 37 C in M9 minimal medium containing 15 NH 4 Cl and U-13 C glucose. Cells obtained from M9 cultures were disrupted by sonication.…”

Section: Sample Preparationmentioning

confidence: 99%

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Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

Park

Ames

2011

Protein Science

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Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, regulates the Ca 21 -dependent activity of inositol 1,4,5-triphosphate receptors (InsP3Rs) and various voltage-gated Ca 21 channels. Here, we present the NMR structure of fulllength CaBP1 with Ca 21 bound at the first, third, and fourth EF-hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root-mean-squared deviation of 0.54 Å (N-domain) and 0.48 Å (C-domain). The first 18 residues from the N-terminus in CaBP1 (located upstream of the first EFhand) are structurally disordered and solvent exposed. The Ca 21 -saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N-domain structure of CaBP1 contains two EF-hands (EF1 and EF2), both in a closed conformation [interhelical angles 5 129°(EF1) and 142°(EF2)]. The C-domain contains EF3 and EF4 in the familiar Ca 21 -bound open conformation [interhelical angles 5 105°( EF3) and 91°(EF4)]. Surprisingly, the N-domain adopts the same closed conformation in the presence or absence of Ca 21 bound at EF1. The Ca 21 -bound closed conformation of EF1 is reminiscent of Ca 21 -bound EF-hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca 21 -bound closed conformation of EF1 in CaBP1 might undergo an induced-fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.

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Section: Sample Preparationmentioning

confidence: 99%

“…23 The first 18 residues from the N-terminus exhibited weak NMR signals that could not be assigned. The remaining residues (19-167) exhibited strong NMR peaks, whose sequence-specific assignments were described previously 27 (BMRB No.…”

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confidence: 99%

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

Park

Ames

2011

Protein Science

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“…This results in a constitutively closed conformation of the aminoterminal domain, whereas the carboxy-terminal domain can switch to an open conformation upon Ca 2þ binding to EF-3 and EF-4. Comparison of the carboxy-terminal domain with that of calmodulin, however, still reveals differences in exposed hydrophobic residues thought to mediate target interactions (Wingard et al 2005).…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

“…Analogy to calmodulin would suggest that the CaBPs should adopt a dumbbell like tertiary conformation consisting of an aminoterminal domain containing EF-1 and EF-2 and a carboxy-terminal domain containing EF-3 and EF-4, connected by a central linker. NMR analysis revealed that CaBP1 does indeed have two independent, noninteracting domains, joined by a flexible linker (Wingard et al 2005 (Wingard et al 2005;Li et al 2009). The Mg þþ bound form of CaBP1 is similar to that of apo-calmodulin, but the Ca 2þ bound form appears markedly different.…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

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The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

McCue

Haynes²,

Burgoyne

2010

Cold Spring Harbor Perspectives in Biology

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Conformational dynamics of recoverin's Ca²⁺‐myristoyl switch probed by ¹⁵N NMR relaxation dispersion and chemical shift analysis

Ishima

Ames

2011

Proteins

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Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca 2+ -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca 2+ -myristoyl switch. Here we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on 15 N-labeled recoverin to probe main chain conformational dynamics. 15 N NMR relaxation data suggest that Ca 2+ -free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca 2+ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca 2+ -dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca 2+ saturated extruded state (R): T ↔ I ↔ R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ ex δω < 1). The final step (I ↔ R) is much slower (τ ex δω > 1). The main chain structure of I is similar in part to the structure of half-saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ↔ I may transiently increase the exposure of Ca 2+ -binding sites to initiate Ca 2+ binding that drives extrusion of the myristoyl group during I ↔ R.

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Structural Analysis of Mg2+ and Ca2+ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels

Cited by 80 publications

References 64 publications

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Conformational dynamics of recoverin's Ca²⁺‐myristoyl switch probed by ¹⁵N NMR relaxation dispersion and chemical shift analysis

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Structural Analysis of Mg2+ and Ca2+ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels

Cited by 80 publications

References 64 publications

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca2+‐bound closed state: Implications for target recognition

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca2+‐bound closed state: Implications for target recognition

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Conformational dynamics of recoverin's Ca2+‐myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

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Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

Nuclear magnetic resonance structure of calcium‐binding protein 1 in a Ca²⁺‐bound closed state: Implications for target recognition

Conformational dynamics of recoverin's Ca²⁺‐myristoyl switch probed by ¹⁵N NMR relaxation dispersion and chemical shift analysis