Abstract
A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.
Highlights
3083, Australia. 2 Both authors contributed to this work. 3 Both authors made equal senior author contributions to this work. 4 To whom correspondence should be addressed
We found that tethering the ends of the II-III loop increased ␣-helical and -strand structure in particular regions of the loop involved in key protein-protein interactions and excitation contraction (EC) coupling
We find that cyclization of the skeletal dihydropyridine receptor (DHPR) IIIII loop affects the secondary structure and the dynamic properties of the helical A/B region as well as the critical C region
Summary
3083, Australia. 2 Both authors contributed to this work. 3 Both authors made equal senior author contributions to this work. 4 To whom correspondence should be addressed. To more closely mimic the tethered nature of the II-III loop N and C termini in the intact membrane-bound protein and the in vivo situation, we have used an intein-based method to link the N- and C- terminal ends of the loop. We found that tethering the ends of the II-III loop increased ␣-helical and -strand structure in particular regions of the loop involved in key protein-protein interactions and EC coupling. This structural change strikingly increased the affinity of the II-III loop for the SPRY2 domain and enhanced its ability to activate RyR1 channels in lipid bilayers
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