Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: implications for target recognition

Abstract

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, regulates the Ca(2+) -dependent activity of inositol 1,4,5-triphosphate receptors (InsP3Rs) and various voltage-gated Ca(2+) channels. Here, we present the NMR structure of full-length CaBP1 with Ca(2+) 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 EF-hand) are structurally disordered and solvent exposed. The Ca(2+) -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 = 129° (EF1) and 142° (EF2)]. The C-domain contains EF3 and EF4 in the familiar Ca(2+) -bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N-domain adopts the same closed conformation in the presence or absence of Ca(2+) bound at EF1. The Ca(2+) -bound closed conformation of EF1 is reminiscent of Ca(2+) -bound EF-hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca(2+) -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.

Figure 1

¹⁵N–¹H HSQC spectra of Ca²⁺-saturated CaBP1 for the N-domain fragment (residues 1–91) (A) and C-domain fragment (residues 96–167) (B).

Figure 2

¹⁵N NMR relaxation data showing {¹H}–¹⁵N NOE (A), ¹⁵N longitudinal relaxation rate (B), and ¹⁵N transverse relaxation rate (C) for Ca²⁺-saturated CaBP1. A schematic representation of the secondary structure is shown at the top with α-helices and β-strands indicated by boxes and arrows, respectively.

Figure 3

Correlation between experimentally measured RDC values and back-calculated RDC values from Ca²⁺ bound CaBP1 N-domain (A) and C-domain (B). The NH RDCs used for structure calculation (47/23 RDCs), the subset of NH RDCs withheld from the structure calculation (6/6 RDCs), and C′−Ca RDCs (30/24 RDCs) were all used to calculate the overall Q-factor and R-factor. For the cross-validation, the subset of NH RDCs (6/6 RDCs) and C′–Ca RDCs (30/24 RDCs) was used to calculate the free Q-factor and R-factor. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Main chain structures of Ca²⁺-saturated CaBP1 determined by NMR. Superposition of the 15 lowest energy structures (A) and ribbon representations of the energy-minimized average structure (B) are illustrated for the N-domain (left) and C-domain (right). N-terminal residues (1–19) are unstructured and not shown. EF-hands are highlighted in color. Orange spheres represent bound Ca²⁺. Protein databank accession nos. 2LAN and 2LAP.

Figure 5

Space-filling representation of Ca²⁺-saturated CaBP1 illustrating the N-domain (top) and C-domain (bottom). Acidic residues (Asp and Glu), basic residues (Arg, His, and Lys), and hydrophobic residues (Ile, Leu, Phe, Met, and Val) are colored red, blue, and yellow, respectively.

Figure 6

Schematic model showing possible structural interactions of CaM and CaBP1 with voltage-gated Ca²⁺ channels during calcium-dependent inhibition (CDI, left panel) and calcium-dependent facilitation (CDF, right panel). The CaBP1 C-domain (gray) and CaM C-domain (green) both compete to interact with the CaV1.2 IQ-motif (yellow) in the C-terminal cytosolic domain. The CaBP1 N-domain (pink) switches from a closed to open conformation upon binding to a separate helical segment in the channel that may play a role in CDF as described by Ref.. Bound Ca²⁺ (orange), N-terminal myristoyl group (red), CaM (green), and transmembrane channel domain (light blue) are highlighted.