LIM domain-wide comprehensive virtual mutagenesis provides structural rationale for cardiomyopathy mutations in CSRP3

Figures

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  Figure 1. CSRP3 HCM/DCM mutations. (A) CSRP3 LIM domains length and mutations mapped to the full protein shows localisation of mutations to the LIM domains. (B) NMR structures of LIMs and mutations mapped on them (blue color spheres) using PDB ID: 2O10 for LIM1 and PDB ID: 2O13 for LIM2. (C) Structural stability analysis of LIM domain HCM (blue color) and DCM (green color) mutations. Severity of mutations are indicated by shades of red color (dark red and pink). Neutral mutations are shown as black. Dotted red and green lines depict the threshold for deciding destabilising and stabilising mutations. (D) Bar plot highlighting the mutational stability of CSRP3 LIM domains. The plot shows the significance of these mutations on ensemble of 19 and 20 NMR conformers of LIM1 and LIM2 domains, respectively.

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  Figure 2. Mutational landscape of CSRP3 LIMs. Mutational severity are categorised based on ∆∆G (kcal/mol) values and plotted as circles. Circle is colored dark red when highly-destabilising (∆∆G > 3), red in case of destabilising (3 ≥ ∆∆G > 2), pink if slightly de-stabilising (2 ≥ ∆∆G > 1), grey when neutral (1 ≥ ∆∆G > − 1), light green when slightly stabilising (− 2 < ∆∆G ≤ − 1), green if stabilising (− 3 < ∆∆G ≤ − 2) and as forest green when highly stabilising (∆∆G ≤ − 3). Original amino acid is kept as blank without any circle. Reported HCM/DCM mutations are marked with yellow star. (A) LIM1 map depicting its mutational flexibility. Leucine 44 substitutions are highlighted in a red color box. (B) LIM2 mutational flexibility map.

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  Figure 3. Cross-correlation between residue pairs of LIM1 domain. Pearson correlation method was used by CONAN tool for WT, L44M and L44P trajectories. Anti-correlated residues pairs are colored in brown shade while correlated residues pairs are shown in blue color shades. Red outlined box indicates L44 region. Green outlined box indicates residue correlation differences between WT, L44M and L44P trajectories.

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  Figure 4. Secondary structural elements and residue interaction analysis of WT, L44M and L44P substitutions. (A) Timeline of secondary structure conformations in the trajectories of WT, L44M and L44P structures. In the representation, ‘T’ (aqua) corresponds to ‘Turn’, ‘E’ (yellow) indicates ‘Extended β-strand conformation’, ‘C’ (white) stands for ‘Coil’ (random coil) and ‘B’ (pea green) depicts ‘isolated bridge’. (B) Interactions in the region of position 44 of LIM1 domain. Hydrophobic (yellow color dash lines) and hydrogen bond (green color dash lines) interactions are highlighted for WT, L44M and L44P structures. (C) The electrostatic potential of WT, L44M and L44P structures, corresponding to zeroth snapshot of the dynamics simulations, are shown as a surface representation. Zn2+ is indicated in yellow sphere and position 44 is highlighted in pink ball and stick form.

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  Figure 5. Sequence map of CSRP3 in representative eukaryotes. (A) Multiple sequence alignment of CSRP3 proteins from selected eukaryotes. (B) Consurf mapping of conserved residues to the structures (left: PDB ID: 2O10; right: PDB ID: 2O13). Blue color shade depict highly variable residue positions (value 1 = highly variable), while dark pink color shade show conserved residues positions (value 9 = extremely conserved). (C) Maximum Likelihood Phylogeny of CSRP3 protein in the representative eukaryotes with 1000 bootstrap iterations (pink circles). Different taxa are colored uniquely (see legend).