We next addressed the possible impact of exon 17b on TLN1 protein function. TLN1 is comprised of 2,541 amino acids, that make up the 18 talin domains (Fig. 3 A). Exon 17b introduces a 17 amino acid, in-frame insertion immediately after residue Gln665 (Fig. 3 B). Gln665 is in the first helix of the R2 bundle (Fig. 3, B–D). The crystal structure of talin R1R2 has been solved, which revealed that the two rod domains pack against each other in a side-to-side arrangement (Fig. 3 C; Papagrigoriou et al., 2004). To establish the effect of 17b on this region, we generated structural models of both R1R2-WT and the R1R2-17b proteins using the protein structure prediction tool AlphaFold (Senior et al., 2020). Both the R1R2-WT and R1R2-17b structural models (Fig. S1 E) showed good agreement with the crystal structure of the wild-type R1R2 (Papagrigoriou et al., 2004) validating that the models were accurate. The R1R2-17b structural model showed that the 17 amino acid insert extends the linker region between R1 and R2 (Fig. 3, E and F) and is predominantly unstructured except for a small helical region in the linker (Fig. 3 F), this helical region is from residues that were originally part of R2 before the insertion. The model also predicts structural differences within both the R1 and R2 domains. With insertion of the 17 aa sequence, the first helix of R2 is one turn shorter because the linker now incorporates the first three residues Pro662-Gln665, from the helix. Furthermore, in the wild-type R1R2 structure the linker between R1 and R2 is short, tightly linking the end of R2 with the start of R1 (Fig. 3 D). The extended linker relieves this conformational constraint so that the last helix of R1 is extended by three residues (E657, S658, D659; Fig. 3 F). Interestingly, this extension comes from residues from in the R1R2 linker (blue) that, with the increased linker length, become part of the fifth helix of R1. It seems reasonable to assume that the helix could be extended in a similar fashion in WT-TLN1 if R2 was to unfold.

Full-length TLN1 can adopt open (Fig. 3 A) and autoinhibited (Fig. 3 G) states; overlaying the structural model of R1R2-17b on the cryo-EM structure of autoinhibited monomeric TLN1 (Dedden et al., 2019) shows that the 17b insertion is exposed on the surface of the closed conformation. In the context of the 250 kD full-length molecule, these additional 17 residues will be hard to detect by SDS-PAGE, which probably contributed to its late identification. In the context of R1R2 alone, the splice variant is readily detectible by SDS-PAGE due to the additional ∼2 kD (Fig. 3 H). Together, these data suggest that the 17b insertion may perturb the mechanical behavior and function of the R1R2 region of talin1.