Structural characterisation of the IIICS-containing region of fibronectin: Splice variants define propensity for long-range interactions.

Fibronectin (FN) is an abundant glycoprotein of the extracellular matrix (ECM), interacting with cell-surface integrins and proteoglycans to promote cellular adhesion, migration, and differentiation. The accessibility of FN's cell-binding motifs is tightly regulated by a system of intramolecular FN interactions and alternative splicing, allowing tissue and context-specific activity. The type III connecting segment (IIICS) of FN undergoes complex alternative splicing of its A, B, and C subsegments, with the IIICS-A-containing isoforms harbouring the pro-metastatic α4β1 integrin-binding motif. Despite extensive investigation of IIICS-mediated functions, the structural properties of the IIICS and its influence on neighbouring FN domains remain poorly understood. Here, we use solution-state NMR spectroscopy to characterise the structure and dynamics of the IIICS-containing region across multiple splice isoforms. We show that the IIICS is an intrinsically disordered region (IDR), displaying different levels of disorder between its N- and C-terminal halves, independent of splicing boundaries. The IIICS is also found to undergo spontaneous deamidation, in common with other regions of FN, but at a distinct motif, TGNG. While the IIICS does not contribute to the structural scaffold of the adjacent FNIII15 repeat, which adopts a non-canonical six-stranded fold, its presence promotes long-range intramolecular interactions involving FNIII15. Importantly, the strength of interaction is modulated by IIICS splicing, with interactions greatest when the IIICS-C subsegment is absent. Our results reveal that, despite being intrinsically disordered, the IIICS exhibits non-random-coil behaviour, with region-specific dynamics and conformational sampling. This non-random behaviour facilitates the formation of previously unidentified FN-FN interactions, which we propose contribute to FN's compact conformation and the burial of cryptic binding sites.