Chemical shift assignments of calmodulin bound to the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Abstract

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca²⁺-bound CaM, which promotes Ca²⁺-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca²⁺-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

Keywords: CNGB1; CaM; Calcium; NMR; Photoreceptor; Retina.

Fig. 1

Two-dimensional NMR spectra of CaM bound to unlabeled CNGB1-CaMBD peptide. A ¹⁵N–¹ H HSQC spectrum recorded at 600 MHz ¹H frequency was analyzed to determine backbone resonance assignments. B Expanded view of resonance assignments from the spectrally crowded region highlighted by the dashed box. C Constant-time ¹³C–¹H HSQC spectrum was analyzed to determine side chain resonance assignments. Representative resonance assignments are indicated by residue labels; complete assignments are available as BMRB accession no. 51222

Fig. 2

Secondary structure and order parameters of Ca²⁺-saturated CaM bound to unlabeled CNGB1 peptide predicted from the assigned backbone chemical shifts. A Probability of secondary structural elements (cyan for helix and magenta for strand) and B RCI order parameter (RCI-S²) of Ca²⁺-saturated CaM bound to unlabeled CNGB1 peptide were predicted using TALOS+ server (Shen et al. 2009). The wire diagram depicting the secondary structural elements (cylinder for helix and triangle for strand) was obtained from the CaM structure [PDB ID—2VAY (Halling et al. 2009)]

Fig. 3

Residue-specific amide chemical shift perturbation (CSP) for Ca²⁺-bound CaM in the presence and absence of CNGB1 peptide. CSP was calculated as: $CSP=\sqrt{{\left(\mathrm{\Delta },H^N\right)}^2+{\left(\mathrm{\Delta },N\right)}^2}$. ΔH^N and ΔN are the observed difference in the ¹H^N and ¹⁵N chemical shifts, respectively for CaM/CNGB1 compared to CaM alone. CSP values are mapped on to the CaM structure (PDB ID: 2VAY (Halling et al. 2009))