009-23 – Autosomal Dominant Frontal Lobe Epilepsy-associated variant G155D affects the structure-function relationship of Calcium Binding Protein 4

Introduction: Autosomal dominant frontal lobe epilepsy (ADNFLE) is a partial epilepsy disorder resulting in frequent focal seizures. ADNFLE patients (~30%) currently do not respond to treatment. Calcium binding Protein 4 (CaBP4) modulates the activity of the voltage-dependent calcium channel Cav1.4. Cav1.4 is mainly expressed in photoreceptor synaptic terminals and is key for maximal glutamate release in low light conditions. The point mutation G155D in CaBP4 has been linked to ADNFLE, however the effect of this mutation on the structure-function relationship of CaBP4 is still unknown.
Methods: Using computational biology (AlphaFold and 2Struc) and circular dichroism (CD), we investigated the alterations caused by the G155D mutation to the secondary structure. Changes to the stability of the protein were investigated through computational predictions (DynaMut), susceptibility to protease digestion (SDS-PAGE) and thermostability (CD). The effect of G155D on CaBP4 affinity for calcium was determined using intrinsic fluorescence spectroscopy (tyrosine).
Results and Conclusions: In both calcium-bound and calcium-free conditions, the G155D variant showed 10 +/- 1% decrease in alpha-helical content and 5 +/- 1% increase in unordered structure. DynaMut predictions illustrated a significant gain in flexibility in the region of the G155D mutation and an increased number of intra-molecular interactions. Stability investigations highlighted that the G155D mutation destabilised CaBP4 structure with a higher susceptibility to protease digestion and significantly decreased thermostability (melting temperature decreased from 44oC to 38oC, when compared to wild-type). Equilibrium calcium binding titrations showed a significant decrease in calcium affinity for the G155D variant. In summary, we demonstrated that the ADNFLE-associated mutant G155D significantly affects structure, stability and calcium affinity of CaBP4.