title: Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1areveal critical roles of domain interactions for stability.
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 α1and 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 β1aRemoval of the intrinsically disordered N and C termini and the hook region of β1aprevented 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 β1aand β2aconstructs, and the addition of the DHPR α1sI-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6-8 °C. Similar to other β isoforms, β1abound 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 β1awhile also providing a conduit for allosteric signaling events.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Norris
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Soumya
authors.initials: S
authors.lastname: Joseph
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Shouvik
authors.initials: S
authors.lastname: Aditya
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Yamuna
authors.initials: Y
authors.lastname: Karunasekara
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.affiliation: Department of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Oakley
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia. Electronic address: Marco.Casarotto@anu.edu.au.
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D051528:Guanylate Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: C492923:Cacnb1 protein, mouse; D020746:Calcium Channels, L-Type; D051528:Guanylate Kinases
keywords: X-ray crystallography; dihydropyridine receptor (DHPR); excitation-contraction coupling (E-C coupling); protein structure; skeletal muscle