Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation

Fig. 1. Crystal structures of titin Fn3-3, Fn3-11, Fn3-20, Fn3-49, Fn3-56, Fn3-85 and Fn3-90. A: Diagram showing main components of striated muscle half-sarcomere and domain structure of A-band region of Titin. Domain position of the crystal structures solved and the variants studied in this work are indicated. B: Diagram of Titin A-band D-zone and C-zone super-repeat domain pattern. Position of the studied domains in these super-repeats are indicated. C: Crystal structures of wild type Titin domains solved in this work shown in cartoon and surface representation. All structures are presented in the same orientation with their N- and C- termini on the left and right, respectively. Sidechains of residues conserved in all titin Fn3 domains are shown in a stick representation. Sidechains of residues with variants studied in this work are coloured green and shown in a stick representation. D: Structural sequence alignment of domains in this work with the following residue colour scheme: positively charged, magenta; negatively charged, blue; small and/or hydrophobic, red; polar and glycine, green. Conserved (*), strongly conserved (:) and weakly conserved (.) residues as defined in Clustal…

Fig. 2. Comparison of WT and variant Fn3-3 (left) and Fn3-56 (right) domain crystal structures. A: cartoon representation of aligned WT and variant domain structures, with mutated residue sidechains shown in stick representation. B: detail of region surrounding mutated residue. Sidechains shown in stick representation. C and D: detail of electron density surrounding WT (C) residue mutated in variant structure (D). 2Fo-Fc maps displayed at sigma = 1.0.

Fig. 3. Position of residues in crystal structures of Titin Fn3-11, Fn3-85 and Fn3-90 mutated in patients with hypertrophic cardiomyopathy. Ala16872Pro, Ser27083Pro and Arg27839Gln were identified in patients from HCM cohorts. Residue mutated in patients highlighted in green, polar contacts shown as dashed yellow lines. Residues of interest shown in stick representation.

Fig. 4. Biophysical analysis of purified titin domains with variants from HCM patients. A: analytical gel filtration traces of Fn3-11 wt and Ala16872Pro. B: 1D NMR of Fn3-11 wt and Ala16872Pro. C: 1D NMR of Fn3-85 wt and Ser27083Pro. D: 1D NMR of Fn3-90 wt and Arg27839Gln. E: differential scanning fluorimetry (DSF) plots showing unfolding of WT Fn3-3, Fn3-56, Fn3-85 and Fn3-90 and their HCM-linked variants Arg15908His, Ser23226Gly, Ser27083Pro and Arg27839Gln, respectively. F: Melting temperatures, Tm, of each domain calculated from DSF unfolding curves. Statistical difference between the means of WT and variant melting temperatures calculated using the student’s t-test. n = 3–5 technical repeats, **** = P < 0.0001.

Fig. 5. Position of residues in crystal structures of Titin Fn3-20, Fn3-49 and Fn3-90 mutated in congenital myopathy patients. Leucine 18,237 mutated to proline in Fn3-20, valine 22,232 mutated to glutamic acid in Fn3-49 and glycine 27,849 mutated to valine in Fn3-90 in patients with congenital myopathy, co-inherited with either one truncating variant in trans (Fn3-49 and Fn3-90) or two truncating variants (Fn3-20). Residue mutated in patients highlighted in green, hydrogen bonds shown as dashed yellow lines. Residues of interest shown in stick representation.

Fig. 6. Assessment of domain-domain interactions within crystal. A: For each determined WT crystal structure, the homo-dimeric interaction with the largest surface area is presented, with the relative orientation of each protein chain indicated by the arrow pointing towards the C-terminus. B: alignment of the domain sequences, with the residues involved in the interacting surface in (arbitrary) chain A boxed in green, chain B boxed in blue and both chains boxed in purple.