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

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 alpha(1) and beta subunits ofDHPR 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 beta(1a). Removal of the intrinsically disordered N and C termini and the hook region of beta(1a) prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined beta 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 similar to 2-3 degrees C between the beta(1a) and beta(2a) constructs, and the addition of the DHPR alpha(1s) I-II loop (alpha -interaction domain) peptide stabilized both beta isoforms by similar to 6-8 degrees C. Similar to other beta isoforms, beta(1a) bound with nanomolar affinity to the alpha-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 beta(1a) while also providing a conduit for allosteric signaling events.