Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1)

Abstract

Calmodulin (CaM) plays a vital role in calcium homeostasis by allosterically modulating intracellular calcium channels including the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1). Apo (calcium-free) CaM activates hRyR1 while calcium-saturated CaM inhibits it. Two CaM-binding regions (residues 1975–1999 and 3614–3643) identified in each RyR1 monomer were proposed to allow CaM to bridge adjacent RyR1 subunits. We explored the distinct roles of CaM domains by using fluorescence anisotropy to determine the affinity of CaM1–148 (full-length), CaM1–80 (N-domain) and CaM76–148 (C-domain) for peptides encompassing hRyR1 residues 1975–1999 or 3614–3643. Both CaM1–148 and CaM76–148 associated in a calcium-independent manner with similar affinities for hRyR1(3614–3643)p while CaM1–80 required calcium and bound ~250-fold more weakly. Association of CaM1–148, CaM1–80 and CaM76–148 with hRyR1(1975–1999)p was much less favorable than with hRyR1(3614–3643)p; differences between the two CaM domains were smaller. Equilibrium calcium titrations monitored by steady-state fluorescence demonstrated that both hRyR1 peptides increased the calcium-binding affinity of both CaM domains. These thermodynamic properties support a prior model in which the CaM C-domain associates with RyR1(3614–3643) at low levels of calcium, positioning CaM to rapidly respond to calcium efflux. However, the affinity of the N-domain of CaM for hRyR1(1975–1999)p is insufficient to explain a model in which CaM bridges adjacent RyR1 subunits within the tetramer. This indicates that other protein factors or properties of the tertiary or quaternary structure of hRyR1 contribute to the energetics of CaM-mediated regulation.

Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin

“…CaM binding properties of the RyRs have focused on RyR1. It has been reported that apoCaM and Ca 2þ -CaM bind with nanomolar affinity (similar to that of the full-length channel) to a synthetic peptide matching amino acids 3614-3643 of RyR1 (RyRp) (11,12), while other studies suggest that the affinity is much lower (micromolar) in the apo-state (13,14). In this article, the position number in the 31-residue RyRp starts from 1 at the N-terminus (3614 in full-length receptor) to 31 at the C-terminus (RyRp sequence in Materials and Methods).…”

“…Trypsin cleavage, alkylation (11), and site-directed mutagenesis (16) suggested that apoCaM and Ca 2þ -CaM bind to the same RyR1 region that includes Cys 22 (17) and Leu 11 in RyRp (16). A recent fluorescence anisotropy study proposed a model in which the C-domain of CaM is bound to the RyRp site in the presence and absence of Ca 2þ , but the N-domain of CaM is bound only in the presence of Ca 2þ (13). There is currently limited information on the structure and dynamics of the isolated RyRp, the apoCaM/RyRp complex, and the Ca 2þ -CaM/ RyRp complex in solution.…”

Effects Of Postactivation Potentiation On Subsequent 40-yard Sprint Performance In 16- To 23-year-old Male Athletes

“…Ca 2+ binds to the regulatory protein troponin-C, which causes a conformational shift of the tropomyosin from myosin binding sites on the actin molecule (Frontera & Ochala, 2015;Rebbeck et al, 2014) ii. Ca 2+ plays a role in myosin phosphorylation and conformational activation of calmodulin Newman, Sorensen, Kilpatrick, & Shea, 2014;Rebbeck et al, 2014). This myogenic mechanism will be further discussed later in this review.…”

“…The ELCs are located closer to the N-terminal, acting as a stabilization mechanism for the N-terminal of the myosin molecule, and the RLCs are the phosphorylatable light chains, located closer to the filamentous backbone of the myosin molecule ( Figure 2); (Gardiner, 2001). Specific enzymes enable conformational alterations of the positioning of the myosin N-terminal during prolonged bouts of muscular activation Midde et al, 2013;Newman et al, 2014). Figure 2: Ca2+ / CaM interaction with MLCK, shows the conformational shift in CaM when the Ca 2+ -binding sites are occupied by Ca 2+ .…”

“…This conformational shift leaves the N-terminal of the myosin molecule to mechanically transition away from the myosin's backbone and towards actin molecules (Figure 4). The conformational shift engenders an environment that is advantageous to actin-myosin interaction and cross-bridge formation due to the increased proximity of the actin and myosin molecules (Brooks et al, 2005;Gardiner, 2001;Newman et al, 2014). Thus, skMLCK phosphorylation indirectly promotes increases in crossbridge formation by increasing the likelihood of actin-myosin interaction.…”