%0 Journal Article
%T Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability.
%A Norris NC
%A Joseph S
%A Aditya S
%A Karunasekara Y
%A Board PG
%A Dulhunty AF
%A Oakley AJ
%A Casarotto MG
%J J Biol Chem
%V 292
%N 20
%D 05 2017 19
%M 28351836
暂无%R 10.1074/jbc.M116.763896
%X 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.