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Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

Bhattacharya D, Mehle A, Kamp TJ, Balijepalli RC - PLoS ONE (2015)

Bottom Line: The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable.Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP.The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.

ABSTRACT
The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

No MeSH data available.


Model of H458 CCPGCC ß1a- CFP in a FRET competent state.The ß1a subunit structure was predicted with Phyre2 and obtained using ß3 subunit as a template. The lower FRET between excitation pairs at H458 (CCGPCC) and the C-terminus (CFP) in the presence of RyR1 suggests a structural change wherein the C-terminal region of the modeled ß1a assumes an extended conformation.
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pone.0131399.g005: Model of H458 CCPGCC ß1a- CFP in a FRET competent state.The ß1a subunit structure was predicted with Phyre2 and obtained using ß3 subunit as a template. The lower FRET between excitation pairs at H458 (CCGPCC) and the C-terminus (CFP) in the presence of RyR1 suggests a structural change wherein the C-terminal region of the modeled ß1a assumes an extended conformation.

Mentions: X-ray crystallographic studies defined the structure of the central core of ß2, ß3 and ß4 subunits revealing interlinked SH3 and GK domains representative of members of the MAGUK superfamily of adapter proteins [29–31]. A less ordered domain in between the SH3 and GK domains composed of the variable central domain of ß subunits is comparable to the ‘hook’ domain described for the related MAGUK protein PSD95, which may be important for interacting with other proteins [51]. The organization of the SH3 and GK domains in the ß subunits takes a more elongated conformation compared to the more compact relationship of these domains in PSD95[31]. Neither the N- nor C-terminal sequences were resolved in these structures and are likely disordered. Despite the fact that the ß1 subunit structure has not been solved, extensive sequence homology between all ß subunit core regions (> 60% identity between ß1 and the other subunits) suggests a comparable structural organization. This identity was exploited to model the structure of the ß1a subunit (Fig 5). The model maintains the core domains and properly positions conserved residues at the binding site for the œ interaction domain (AID). Notably, this model also suggests that H458, a CCPGCC insertion site, is the last residue preceding the flexible C-terminus. Thus, the present intramolecular ex vivo FRET data for the ß1a subunit can reasonably be compared to the solved crystallographic structures for the ß subunits.


Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

Bhattacharya D, Mehle A, Kamp TJ, Balijepalli RC - PLoS ONE (2015)

Model of H458 CCPGCC ß1a- CFP in a FRET competent state.The ß1a subunit structure was predicted with Phyre2 and obtained using ß3 subunit as a template. The lower FRET between excitation pairs at H458 (CCGPCC) and the C-terminus (CFP) in the presence of RyR1 suggests a structural change wherein the C-terminal region of the modeled ß1a assumes an extended conformation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482598&req=5

pone.0131399.g005: Model of H458 CCPGCC ß1a- CFP in a FRET competent state.The ß1a subunit structure was predicted with Phyre2 and obtained using ß3 subunit as a template. The lower FRET between excitation pairs at H458 (CCGPCC) and the C-terminus (CFP) in the presence of RyR1 suggests a structural change wherein the C-terminal region of the modeled ß1a assumes an extended conformation.
Mentions: X-ray crystallographic studies defined the structure of the central core of ß2, ß3 and ß4 subunits revealing interlinked SH3 and GK domains representative of members of the MAGUK superfamily of adapter proteins [29–31]. A less ordered domain in between the SH3 and GK domains composed of the variable central domain of ß subunits is comparable to the ‘hook’ domain described for the related MAGUK protein PSD95, which may be important for interacting with other proteins [51]. The organization of the SH3 and GK domains in the ß subunits takes a more elongated conformation compared to the more compact relationship of these domains in PSD95[31]. Neither the N- nor C-terminal sequences were resolved in these structures and are likely disordered. Despite the fact that the ß1 subunit structure has not been solved, extensive sequence homology between all ß subunit core regions (> 60% identity between ß1 and the other subunits) suggests a comparable structural organization. This identity was exploited to model the structure of the ß1a subunit (Fig 5). The model maintains the core domains and properly positions conserved residues at the binding site for the œ interaction domain (AID). Notably, this model also suggests that H458, a CCPGCC insertion site, is the last residue preceding the flexible C-terminus. Thus, the present intramolecular ex vivo FRET data for the ß1a subunit can reasonably be compared to the solved crystallographic structures for the ß subunits.

Bottom Line: The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable.Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP.The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.

ABSTRACT
The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

No MeSH data available.