Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal 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 (RyR1) Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1s 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 beta 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 (GK) domains. However, transition melting temperatures derived from the DSF 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 (AID) peptide stabilized both beta isoforms by ~ 6−8°C. Similar to other beta isoforms, β1a bound with nanomolar affinity to AID, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and GK domains play a role in the stability of β1a, while also providing a conduit for allosteric signaling events.