Figure 3.
In silico modeling of ITPR3 variants. (A) ITPR3 secondary structure with domains annotated. The location of the AAs affected by dominant variants are marked by pink circles (this study i.e., A196 in P1, I2506 in P2, and R2524 in P3–P5); and yellow circles (other studies [Neumann et al., 2023; Rönkkö et al., 2020; Terry et al., 2022]: V615, T1424, F1628, and R2524); the R1850Q polymorphism is marked by a grey circle. Missense and nonsense variants in gnomAD database are indicated by blue and red vertical lines, respectively. (B) ITPR3 tertiary structure with domains colored as indicated in panel A. The center shows the monomer of the cryo-EM structure 6DRC colored by domains as predicted by Pfam, with minor corrections in the ranges, namely coupling BTF (AA 3–230), IP3 binding BTF (AA 233–433), first IP3 binding helix bundle (437–707), second IP3 binding helix bundle (1175–1334), coupling helix bundle (1335–1546), central helix bundle (1864–1974), and ion channel transmembrane domain (2191–2537). The latter contains, within its S6 helix, a cationic selectivity filter (formed by N2472 and D2478), which favors the passage of Ca2+ over other cations, and a gate (formed by I2517 and F2513) to prevent ions from passing unless the channel is activated (Paknejad and Hite, 2018). The three insets (right) show the overlay of the WT (turquoise) and the variant (salmon) chains with other chains shown in white. (C) Multiple sequence alignment of AA sequences in orthologous ITPR proteins across different species, with the inferred phylogenetic trees displayed on the left-hand side of the panel. AA residues altered in P1–P5 are annotated at the top of the figure and marked by red boxes. (D) Boxplot of transcriptomic distance to closest gnomAD variant for the de novo variants in P1–5 (blue) and gnomAD control variants (grey); *P = 0.02, Wilcoxon rank sum test. (E) Heatmap showing ΔΔG, a calculated parameter that predicts the effect of a protein change on stability, for selected mildly destabilizing ITPR3 variants across five different conformational models of ITPR3. A positive score (red) predicts that a variant is destabilizing, whilst a negative score (blue) predicts a variant to be stabilizing. Variants shown include those identified in P1–P5 of this study; V615M, a pathogenic dominant variant identified in familial CMT disease (Rönkkö et al., 2020); W168A, an in vitro generated variant that abolishes channel activity (Chan et al., 2010; Yamazaki et al., 2010); F1628L, a variant reported in a patient with later onset, milder immune deficiency (Neumann et al., 2023); P187S and I184S, two gnomAD control variants close to the variant detected in P1; and R2471H and I2511V, two further gnomAD control variants within the TMD close to the variants detected in P2–P5.

In silico modeling of ITPR3 variants. (A) ITPR3 secondary structure with domains annotated. The location of the AAs affected by dominant variants are marked by pink circles (this study i.e., A196 in P1, I2506 in P2, and R2524 in P3–P5); and yellow circles (other studies [Neumann et al., 2023; Rönkkö et al., 2020; Terry et al., 2022]: V615, T1424, F1628, and R2524); the R1850Q polymorphism is marked by a grey circle. Missense and nonsense variants in gnomAD database are indicated by blue and red vertical lines, respectively. (B) ITPR3 tertiary structure with domains colored as indicated in panel A. The center shows the monomer of the cryo-EM structure 6DRC colored by domains as predicted by Pfam, with minor corrections in the ranges, namely coupling BTF (AA 3–230), IP3 binding BTF (AA 233–433), first IP3 binding helix bundle (437–707), second IP3 binding helix bundle (1175–1334), coupling helix bundle (1335–1546), central helix bundle (1864–1974), and ion channel transmembrane domain (2191–2537). The latter contains, within its S6 helix, a cationic selectivity filter (formed by N2472 and D2478), which favors the passage of Ca2+ over other cations, and a gate (formed by I2517 and F2513) to prevent ions from passing unless the channel is activated (Paknejad and Hite, 2018). The three insets (right) show the overlay of the WT (turquoise) and the variant (salmon) chains with other chains shown in white. (C) Multiple sequence alignment of AA sequences in orthologous ITPR proteins across different species, with the inferred phylogenetic trees displayed on the left-hand side of the panel. AA residues altered in P1–P5 are annotated at the top of the figure and marked by red boxes. (D) Boxplot of transcriptomic distance to closest gnomAD variant for the de novo variants in P1–5 (blue) and gnomAD control variants (grey); *P = 0.02, Wilcoxon rank sum test. (E) Heatmap showing ΔΔG, a calculated parameter that predicts the effect of a protein change on stability, for selected mildly destabilizing ITPR3 variants across five different conformational models of ITPR3. A positive score (red) predicts that a variant is destabilizing, whilst a negative score (blue) predicts a variant to be stabilizing. Variants shown include those identified in P1–P5 of this study; V615M, a pathogenic dominant variant identified in familial CMT disease (Rönkkö et al., 2020); W168A, an in vitro generated variant that abolishes channel activity (Chan et al., 2010; Yamazaki et al., 2010); F1628L, a variant reported in a patient with later onset, milder immune deficiency (Neumann et al., 2023); P187S and I184S, two gnomAD control variants close to the variant detected in P1; and R2471H and I2511V, two further gnomAD control variants within the TMD close to the variants detected in P2–P5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal