title: Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function.
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.
authors.affiliation: John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Han-Shen
authors.initials: HS
authors.lastname: Tae
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Cui
authors.forename: Yamuna
authors.initials: Y
authors.lastname: Karunasekara
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003500:Cyclization; D053488:DnaB Helicases / Q000737:chemistry / Q000378:metabolism; D047668:Inteins / Q000235:genetics; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000235:genetics; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology
chemical_list: D020746:Calcium Channels, L-Type; D008051:Lipid Bilayers; D021122:Protein Subunits; D019837:Ryanodine Receptor Calcium Release Channel; D053488:DnaB Helicases