Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains

Figures

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  Figure 1.. Overview of CaMBDs of RyR. ClustalW protein sequence alignment of the three human RyR isoforms (hRyR1–3). Amino acids that are identical between hRyR1-3 are highlighted in gray and amino acids that are resolved in the cryo-EM structure of rabbit RyR1 (PDB: 3J8H) are underlined. The dots above the alignment indicate amino acid intervals of five according to the RyR2 sequence.

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  Figure 2.. Approximate locations of CaMBDs in the rabbit RyR1 (rRyR1) structure. Ca2+-saturated CaM (purple) bound to RyR1 CaMBD2 (green) (PDB: 2BCX) was juxtaposed with the 3.8 Å resolution cryo-EM structure of the closed-state rabbit RyR1 homotetramer (PDB: 3J8H) [29,32]. One RyR1 monomer is shown in yellow surface representation, and the other three monomers as gray semi-transparent secondary structure schematics. The CaMBDs are not fully resolved, but the nearest resolved N- and C-terminal amino acids are marked by sequence position and color: CaMBD1a in blue, CaMBD1b in red, CaMBD2 in green, and CaMBD3 in orange. The labeled amino acid positions are numbered according to the equivalent human RyR2 positions.

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  Figure 3.. Measurement and analysis of titration curves for the determination of Ca2+-dependent dissociation constants (KD). (A) Simplified measurement and analysis example. Each row in the microtiter plate (A.1) contained a titration series of peptide with decreasing protein concentration (columns 1–24) at one [Ca2+]. Measuring the FA signal from each well in the row gave a titration curve, i.e. the FA signal as a function of the total protein concentration ([Protein]tot, A.2). The KD for the protein binding to peptide at that particular [Ca2+] was obtained by fitting a stoichiometric binding model to the titration curve (A.3). (B) By repeating this fitting procedure for the titration curves from each row (B.1–2), the KD as a function of [Ca2+] was obtained (B.3). Note that the plots in A.3 and B.3 are double-logarithmic.

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  Figure 4.. Example FA signal curves for the titration of RyR2 CaMBD peptides with CaM. The FA signal from the TAMRA-labeled peptide increased with CaM binding (symbols), and the titration curves were fitted to a stoichiometric binding model (solid lines)—see ‘Experimental Procedures’.

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  Figure 5.. Ca2+-dependent affinities of CaM for binding to CaMBD1a, 1b, 2, and 3. The binding model-fitted affinities (KD) are plotted as a function of [Ca2+]. CaMBD color codings correspond to those indicated in Figure 2. Note the double logarithmic axes. Error bars represent the 95% confidence intervals of two (CaMBD1a and -1b ± P) or three (CaMBD2 and -3) replicates.

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  Figure 6.. Ca2+-dependent affinities of CaM N- and C-domains for binding to CaMBD2 and -3. The binding model-fitted affinities (KD) are plotted as a function of [Ca2+]. CaMBD color codings correspond to those indicated in Figure 2. Note the double logarithmic axes. Error bars represent the 95% confidence intervals of two (CaMBD3) or three (CaMBD2) replicates.

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  Figure 7.. Effects of changing CaMBD2 Trp3587 and Phe3603 anchor residues to Ala on CaM binding. Effects of mutating RyR2 CaMBD2 anchor residues on the Ca2+-dependent affinities of (A) full-length CaM and (B) N- and C-domains for binding to CaMBD2. The binding model-fitted affinities (KD) are plotted as a function of [Ca2+]. Note the double logarithmic axes. Error bars represent the 95% confidence intervals of two replicates.

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  Figure 8.. Visualization of Ca2+ binding to CaM/CaMBD complexes. Ca2+-binding curves (normalized FA signal as a function of [Ca2+]) are shown for full-length CaM/CaMBD complexes (A), for isolated CaM domain/CaMBD2 and -3 complexes (B), and for full-length CaM/CaMBD2 (C) or isolated CaM domains/CaMBD2 complexes (D) with CaMBD2 anchor residues changed to Ala. Solid lines show the fit of a generic Hill model for estimating CaM/CaMBD complex affinities for Ca2+ (see Supplementary Table S3).

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  Figure 9.. Proposed binding scheme for CaM regulation of RyR2 with increasing levels of cytosolic Ca2+. RyR2 CaMBD2 is indicated with a green α-helix and Ca2+ with dark spheres. When binding to RyR2, the CaM N-domain is highlighted in red and the C-domain in purple. Free domains are shown in white. At essentially Ca2+-free conditions (3 nM Ca2+, upper row), the CaM C-domain may bind to CaMBD2, involving Trp-3587, or to another RyR2 CaMBD (orange α-helix), such as CaMBD3. However, at resting cytosolic Ca2+ levels (∼100 nM, middle row), a Ca2+-saturated CaM C-domain binds to the CaMBD2 anchor residue, Trp-3587, and the apo-N-domain may interact with another CaMBD or may remain free. At cytosolic Ca2+ levels ∼1 µM and higher (illustrated as >10 µM, lower row), the CaM C-domain remains anchored to CaMBD2 Trp-3587, whereas the N-domain now binds Ca2+ and interacts with another site on RyR2 (e.g. CaMBD3), or clamps down on CaMBD2 Phe-3603.