Conformational plasticity and evolutionary analysis of the myotilin tandem Ig domains

Figures

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  Figure 1. Evolutionary analysis of myotilin. (a) Schematic presentation of the myotilin molecule. N-terminal part of myotilin is indicated by the serine-rich region, comprised of »mutational hotspot« and hydrophobic residues stretch (yellow). C-terminal Ig domains 1 and 2 are coloured blue and red, respectively, followed by the C-terminal tail. Binding sites for various binding partners are noted above schematics. (b) Disorder tendency plot, calculated with the different prediction software, showing that mainly Ig domains present a structured part of the molecule. (c) Conservation plot of every amino acid residue score, calculated from the multiple-sequence alignment. (d) Conservation of the Ig domains of myotilin as calculated by the Consurf-DB server. Below are presented conserved residues, forming an N-terminal cluster. The colouring scheme presents purple residues as conserved and turquoise as varied. *PPxf motif on Ig2 domain is APxf, whereas sequences from other organisms possess typical PPxf motif (Supplementary Data S1).

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  Figure 2. SAXS analysis of myotilin Ig domains. (a) P(r) vs. r plot for MYOTIg1-2, showing bimodal profile, with the Dmax = 9.37 nm. Curve is showing smooth decay to P(r)=0 at the Dmax and good fit to the experimental data presented in the inset, using GNOM program (s = 4πsinθ/λ). (b) Comparison of dimensionless Kratky plots of MYOTIg1-2 with different proteins as an examples of globular protein (BSA), tandem Ig domains of filamin A with compact conformation (Ig20-21), and the same construct with the addition of migfilin peptide (Ig20-21+migfilin), which increases flexibility of the Ig domains. MYOTIg1-2 shows less compact conformation with the degree of flexibility as indicated by Kratky plot broadening and maxima shift and is similar to Ig22-23 of filamin A. Molecular envelopes of the analysed proteins, with the available high-resolution structures are presented above graph. (c) Fit of the calculated rigid body model by CORAL to experimental scattering with goodness of the fit expressed in χ2. Superposition of the rigid body model with the averaged ab initio reconstructed molecular envelope of MYOTIg1-2 is presented in the inset.

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  Figure 3. Flexibility assessment of the tandem Ig domains. (a) Rg distribution plot among 100 best scoring N-state models (N = 1, 2, 3, 4, 5), where 4-state model, that best match the experimental data, is shown in bold. Convergence of the peaks from N-state models, showing three peaks with the maxima at approximately 2.2, 2.6 and 2.8 nm. Overlay of the experimental scattering data with the computed 4-state profile by the MultiFoXS flexibility approach is presented in the inset. Goodness of the fit is expressed in χ2 (b) Ensemble of 4-state top best-scoring structures, superposed to the Ig domain 2 with the corresponding fractions.

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  Figure 4. Molecular dynamics simulations. (a) RMSF plot from the coarse-grained simulation by the CABS-Flex server. Residues with the highest and lowest RMSF values are shown as yellow and green circles, respectively. Residues with the highest and lowest values are presented on the structure of MYOTIg1-2 with the same colouring scheme as in RMSF plot. (b) All atom, 10 ns simulations of the MYOTIg1-2 (wt) and KE->AA mutants. End-to-end distances through the course of simulations are shown, where Ig domains adopt compact conformation, followed by the reversible extension. (c) Presentation of the extended and compact forms of MYOTIg1-2, showing rotation of the highly conserved Glu344 around the main chain and its involvement in the torsion mechanism. Residues Ile375, Pro376 and Ala429 participate in the hydrogen bonding with the Glu344 and Lys343 and are marked with the red dotted circles. Intra-domain hydrogen bonding of linker residues, analysed through the course of simulation is shown in the inset. Negative charged amino acid residues are coloured red, positive blue, hydrophobic grey and water molecules white. (d) End-to-end distance distribution plot, where wt and mutant KE are showing…