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Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability

Journal Article


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Abstract


  • Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β1a. Removal of the intrinsically disordered N and C termini and the hook region of β1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2–3 °C between the β1a and β2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6–8 °C. Similar to other β isoforms, β1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β1a while also providing a conduit for allosteric signaling events.

Authors


  •   Norris, Nicole C. (external author)
  •   Joseph, Soumya (external author)
  •   Aditya, Shouvik (external author)
  •   Karunasekara, Yamuna (external author)
  •   Board, Philip G. (external author)
  •   Dulhunty, Angela (external author)
  •   Oakley, Aaron J.
  •   Casarotto, Marco G. (external author)

Publication Date


  • 2017

Citation


  • Norris, N. C., Joseph, S., Aditya, S., Karunasekara, Y., Board, P. G., Dulhunty, A. F., Oakley, A. J. & Casarotto, M. G. (2017). Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability. Journal of Biological Chemistry, 292 (20), 8401-8411.

Scopus Eid


  • 2-s2.0-85019637978

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=5791&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4753

Number Of Pages


  • 10

Start Page


  • 8401

End Page


  • 8411

Volume


  • 292

Issue


  • 20

Abstract


  • Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β1a. Removal of the intrinsically disordered N and C termini and the hook region of β1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2–3 °C between the β1a and β2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6–8 °C. Similar to other β isoforms, β1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β1a while also providing a conduit for allosteric signaling events.

Authors


  •   Norris, Nicole C. (external author)
  •   Joseph, Soumya (external author)
  •   Aditya, Shouvik (external author)
  •   Karunasekara, Yamuna (external author)
  •   Board, Philip G. (external author)
  •   Dulhunty, Angela (external author)
  •   Oakley, Aaron J.
  •   Casarotto, Marco G. (external author)

Publication Date


  • 2017

Citation


  • Norris, N. C., Joseph, S., Aditya, S., Karunasekara, Y., Board, P. G., Dulhunty, A. F., Oakley, A. J. & Casarotto, M. G. (2017). Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability. Journal of Biological Chemistry, 292 (20), 8401-8411.

Scopus Eid


  • 2-s2.0-85019637978

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=5791&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4753

Number Of Pages


  • 10

Start Page


  • 8401

End Page


  • 8411

Volume


  • 292

Issue


  • 20