A dihydropyridine receptor α1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor

Cui, Yanfang; Tae, Han Shen; Norris, Nicole C.; Karunasekara, Yamuna; Pouliquin, Pierre; Board, Philip G.; Dulhunty, Angela F.; Casarotto, Marco G.

doi:10.1016/j.biocel.2008.08.004

Cited by 49 publications

(71 citation statements)

References 67 publications

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“…Cloning, Expression, and Purification of the Linear and Cyclic II-III Loop-The linear II-III loop was cloned, expressed, and purified as we have previously described (11). For the intein plasmid construction, the pNW1120 vector, which houses the Synechocystis sp.…”

Section: Methodsmentioning

confidence: 99%

“…Protein production was induced by rapidly increasing the incubation temperature to 42°C, and incubation continued for another 4 h. Cultures were then harvested by centrifugation and stored at Ϫ20°C. The protein was purified using a method similar to that described for the linear loop (11 (11). The NMR sample was made up of 100 M 15 N/ 13 C-labeled cyclic II-III loop in 50 mM phosphate, 200 mM KCl buffer at pH 6.5.…”

Section: Methodsmentioning

confidence: 99%

“…The temperature was controlled by a Melcor peltier temperature controller. Fluoresecence binding experiments and analysis of loop binding to the recombinant SPRY2 protein were carried out as described previously (11).…”

Section: ϫmentioning

confidence: 99%

“…intrinsically unstructured protein that consists of several nascent ␣-helical secondary structures and turns (11). The unstructured nature of the linear II-III loop is reflected in its CD profile, where the spectrum displays a distinct minimum at 198 nm and a slight dip at 222 nm ( Fig.…”

Section: Cyclizing the Skeletal Dhpr Ii-iii Loop Promotes Structural mentioning

confidence: 99%

“…However, in vitro structure and function studies of the II-III loop have thus far been performed with loop constructs expressed as a linear protein with free or untethered N-and Cterminal ends. Although the ends of the untethered loop are in close proximity in the three-dimensional structure (11), the geometrical constraints are obviously very different from those experienced in the intact protein, where the N and C termini of the loop are "tethered" to the S6 helix of the second transmembrane repeat and the S1 helix of the third transmembrane repeat, respectively, both of which are inserted in the membrane of the transverse tubule extensions of the fiber surface.…”

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Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

Tae¹,

Cui²,

Karunasekara

et al. 2011

Journal of Biological Chemistry

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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 ␣ 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 ␣-helical and ␤-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.The release of Ca 2ϩ ions from the sarcoplasmic reticulum of skeletal muscle fibers in response to depolarization of the surface membrane, excitation-contraction (EC) 5 coupling, has three essential molecular components. These are the cytoplasmic linker between the second and third transmembrane repeats (the II-III loop) of the ␣ 1S subunit of the skeletal dihydropyridine receptor (skDHPR), the ␤ 1a subunit of the DHPR, and the skeletal muscle type 1 ryanodine receptor (RyR1). It is generally accepted that membrane depolarization leads to the movement of charged residues in the fourth membrane-spanning helix (S4 segment) of the ␣ 1S subunit and that this "charge movement" causes a conformational change in the II-III loop, which is physically transmitted to the RyR1 channel, to open the ion channel and allow Ca 2ϩ ion flow (1). The role of the II-III loop in EC coupling has been extensively probed and confirmed over the past few decades using in vivo chimeric techniques (2-5), and the critical regions and residues involved in this process have been identified (3, 6). Physical interactions between the II-III loop and RyR1 are also seen in in vitro studies, which reveal an increase in the activity of RyR1 channels when they are exposed to the recombinant DHPR II-III loop (7-10). However, in vitro structure and function studies of the II-III loop have thus far been performed with loop constructs expressed as a linear protein with free or untethered N-and Cterminal ends. Although the ends of the untethered loop are in close proximity in the three-dimensional structure (11), the geometrical constraints are obviously very different from those experienced in the intact protein, where the N and C termini of the loop are "tethered" to t...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ϫmentioning

confidence: 99%

Section: Cyclizing the Skeletal Dhpr Ii-iii Loop Promotes Structural mentioning

confidence: 99%

mentioning

confidence: 95%

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Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

Tae¹,

Cui²,

Karunasekara

et al. 2011

Journal of Biological Chemistry

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A guide to the 3D structure of the ryanodine receptor type 1 by cryoEM

Samsó

2016

Protein Science

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Signal transduction by the ryanodine receptor (RyR) is essential in many excitable cells including all striated contractile cells and some types of neurons. While its transmembrane domain is a classic tetrameric, six-transmembrane cation channel, the cytoplasmic domain is uniquely large and complex, hosting a multiplicity of specialized domains. The overall outline and substructure readily recognizable by electron microscopy make RyR a geometrically well-behaved specimen. Hence, for the last two decades, the 3D structural study of the RyR has tracked closely the technological advances in electron microscopy, cryo-electron microscopy (cryoEM), and computerized 3D reconstruction. This review summarizes the progress in the structural determination of RyR by cryoEM and, bearing in mind the leap in resolution provided by the recent implementation of direct electron detection, analyzes the first near-atomic structures of RyR. These reveal a complex orchestration of domains controlling the channel's function, and help to understand how this could break down as a consequence of disease-causing mutations.

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Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation‐contraction coupling

et al. 2022

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Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

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A dihydropyridine receptor α1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor

Cited by 49 publications

References 67 publications

Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

A guide to the 3D structure of the ryanodine receptor type 1 by cryoEM

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation‐contraction coupling

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