Molecular insights into titin’s A-band

Figures

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  Fig. 1. PaSiMap analysis of FnIII-domain sequences from the D- and C- zone of titin. (A) Schematic representation of the organization of domains into super-repeats in the D- and C-zones of A-band titin. FnIII modules are shown in grey and Ig domains in white; as reference the titin kinase domain in the sarcomeric M-line is displayed in black. Super-repeat boundaries are indicated by dividing lines. FnIII domains with previously known atomic structure are shown in yellow. The A84-A86 tandem structurally characterized in this study is boxed; (B) 2D-vector map of pairwise similarities for 117 FnIII domain sequences generated using PaSiMap (Su et al. 2022). Coordinates are colour-coded according to angular spread revealing the existence of three clusters corresponding to three FnIII sequence types. Domains for which 3D-structures are available are labelled; (C) Representation of tandem units of the D- and C-zone, where individual FnIII components are labelled and coloured according to cluster membership in B. Domains for which crystal structures are available are highlighted by a black box. Ig domains are unlabelled and shown as white boxes. (Positional numbering of FnIII domains within the…

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  Fig. 2. Occurrence of distinct systematic differences in A-band domains. (A) PaSiMap sub-analysis of individual FnIII groups as defined by clusters identified in Fig. 1B and A-band Ig mapping as defined in (Su et al. 2022). Sequence points are coloured in a white-to-grey gradient according to vector length from the origin, where dark grey (longest vector) corresponds to prototypic domains with dominant systematic differences and lighter grey (shorter vectors) corresponds to the most variable domains (white is the map origin: coordinates 0,0). (B) D- and C-zone super-repeats where individual domains are coloured as in A. (The degree of shading is relative to each vector map and, thus, can only be quantitatively compared within the same sequence group)

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  Fig. 3. Systematic sequence features correlate with domain position within the tandem. PaSiMap sub-analysis of individual FnIII types (A, B and C) and Ig components of titin’s A-band. In vector maps, sequence coordinates are coloured according to angle using a rainbow spectrum and shading white-to-colour from the origin, with coordinates further from the origin being more colour saturated (white indicates the map origin; coordinates 0,0). For each vector map, corresponding D- and C-zone super-repeats are shown where individual domains are coloured as in maps. (Note: the colour gradient is calculated independently for each domain type and, thus, implies no relation across different types)

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  Fig. 4. Crystal structure of A84-A86 in two extended conformations. (A) Sequence alignment of C-zone tandems of known crystal structure. β-strands are indicated as boxes above the sequence. Loops involved in interactions with linker sequences and, thus, part of domain-domain junctions are boxed in red and blue; (B) Crystal structure of A84-A86 in two extended conformations. The structures have been aligned on their central domain (A85) to better illustrate similarities and differences in the position of the remaining domains. β-sheets are coloured orange and blue (colour-coded as in A) and the DE loop is highlighted in black to ease the visualization of domain torsions. Lateral (left) and longitudinal (right) views are provided; C and D. Interdomain regions of A84-A85 and A85-A86, respectively. Hydrogen bonds are shown as dashed lines. Key residues in conserved loop motifs and constituting the interface between the linker and the subsequent domain are shown as sticks and colour-coded as in A. No direct contacts between domains are observed. (Equivalent interfaces for the remaining molecular copy in the crystallographic asymmetric unit are shown in Fig S7)

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  Fig. 5. Conservation of linker sequences in FnIII tandems of titin’s A-band. Conservation in FnIII-interdomain linker sequences. Linker residues are indicated in bold. Residues identical to those in experimental structures are indicated in red. Close sequence conservation to residues in crystal structures is in orange

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  Fig. 6. Sequence conservation specific to each domain position within the C-zone super-repeat. Top: Residues with highest conservation specific to domain position in the super-repeats C2-C11 are highlighted in orange on the sequences from components of the C4 tandem (A76-A86). Residues that differ from the reference sequence or have a mixed consensus are shown above the sequence; Bottom: Mapping of conserved residues onto structural models. Conserved residues are coloured orange (as above). Ig models were obtained from the AlphaFold database (https://alphafold.ebi.ac.uk/download; entry AF-Q8WZ42-F71-model_v4_14001–154,001 accessed 23rd Nov 2022); A77-A78 corresponds to the crystal structure with PDB code 3LPW; A84-A86 was elucidated in this study and A80-A82 was reconstructed in this study from homology models (obtained from the AlphaFold database as above) structurally aligned to the crystal structure of A84-A86

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  Fig. 7. Revised evolutionary model of titin’s A-band super-repeats. (A) Exon structure of titin’s A-band. Exon numbers are written above the protein domains. Longer exons are coloured to ease visual identification. Protein domains are shadowed in grey to indicate conservation as in Fig. 2. Ig domains are indicated by a thicker box. The four protein residues at the beginning of each exon are given; (B) Fibronectin domains are labelled (A, B, C) and coloured-coded as in Fig. 1. Ig domains are shown as white, non-annotated boxes. Linker sequences are indicated as small triangles between FnIII domains and their consensus sequences given. In (i) to (iii), the DPI motif is proposed as the ancestor linker sequence