Introduction – Autosomal dominant frontal lobe epilepsy (ANDFLE) is a form of partial epilepsy disorder characterised by frequent focal seizures with usual onset at around 10 years of age. Currently, 30 % of ADNFLE patients do not respond to treatment. Calcium Binding Protein 4 (CaBP4) regulates the activity of voltage-dependent calcium channels including Cav1.3 and Cav1.4. Cav1.4 is predominantly expressed in photoreceptor synaptic terminals and plays a crucial role in maximal glutamate release under low light conditions. The point mutation G155D in CaBP4 has been associated with ADNFLE, yet its impact on the structure-function relationship of CaBP4 is still unknown.
Methods – Computational biology tools (AlphaFold and DynaMut) alongside circular dichroism (CD) were utilised to examine the G155D induced modifications to secondary and tertiary structure. Predicted changes to protein stability were also considered using computational tools (DynaMut). Susceptibility to protease digestion (SDS-PAGE) and thermostability (CD) were also used to investigate the impact of G155D on protein stability. The impact of the G155D mutation on CaBP4’s calcium affinity was evaluated through intrinsic fluorescence spectroscopy (tyrosine). Isothermal titration calorimetry (ITC) assessed whether G155D caused changes to CaBP4’s affinity for the IQ domains of voltage-dependent calcium channels (Cav1.2, Cav1.3, Cav1.4).
Results and Conclusions – Under both calcium-bound (n = 5) and calcium-free conditions (n = 5), the G155D variant exhibited a decrease of 10 ± 1% in alpha-helical content and a 5 ± 1% increase in unordered structure. DynaMut predictions revealed a notable increase in flexibility within the region of the G155D mutation and an elevated number of intra-molecular interactions. Susceptibility to protease digestion showed that the G155D mutation destabilised the protein in both calcium-bound (n = 7) and calcium-free conditions (n = 5). The G155D variant also displayed a significant reduction in thermostability with V50 values dropping from 44.0 ±0.8 °c to 37.9 ± 0.5 °c in calcium-free conditions (n = 6) and from 82.5 ± 1.3 °c to 76.2 ± 1.2 °c when calcium-bound (n = 6). Equilibrium calcium titrations showed a 2-fold reduction in Ca2+ affinity (n = 3) for the G155D variant. There was also reduced affinity seen between the G155D protein and all the voltage-dependent calcium channels’ IQ domains. For the Cav1.2 IQ domain there was a 3.6-fold reduction in affinity from Kd CaBP4 = 1.85 M (n = 2) to Kd G155D = 6.59 M (n = 4). Meanwhile for both Cav1.3 and Cav1.4 there was a 4.7 fold reduction in affinity from Kd CaBP4 = 1.85 M (n = 5) to Kd G155D = 8.67 M (n = 7) and from Kd CaBP4 = 1.59 M (n = 5) to Kd G155D = 7.41 M (n = 6) respectively. To summarize, our findings indicate that the ADNFLE-associated mutant G155D has a significant impact on the structure, stability and binding affinity of CaBP4. This study marks the beginning of understanding the mechanism in which the G155D point mutation in CaBP4 leads to ADNFLE.