For the first time, we reveal a direct interaction between Rap1b and the fly homolog of talin, Rhea. Using a combination of biochemical and biophysical techniques, the Rap1 binding site on Rhea has been successfully mapped. Additionally, we reveal that an acidic-to-basic K17E substitution, on Rhea, completely abolishes Rap1 binding. Our collaborators have shown that this mutation results in non-viable embryos and our data links the Rap1:Rhea interaction to this lethal phenotype. The implications of our findings support currently proposed mechanisms of RIAM-independent integrin activation, that would challenge our understanding of focal adhesion formation. \ud \ud Furthermore, we propose a double-dependent Rap1 integrin-activation pathway, involving Rap1 directly interacting with the FERM domain, alongside the known Rap1-dependent recruitment of talin. \ud Optimisations have allowed us to express both the wild-type and mutant Rhea F0 domain in E.coli BL21(DE3) cells. Efficient purification via Ni-NTA-based affinity chromatography results in yields of ~50-60 mg/litre being obtained. Using circular dichroism, it is shown that substitution of the K17 residue does not interfere with the structural integrity of Rhea; both proteins have identical full spectrum measurements and Tm values.\ud \ud Optimal expression of the conserved G-domain of mouse Rap1b was achieved in the CK600K cell line. This region is highly conserved to that in fly (90% identical). NMR was used to show direct interaction between drosophila Rhea F0 and Rap1b; whilst additionally confirming that Rap1b was unable to induce chemical shifts in the F0-K17E mutant. Triple resonance NMR experiments revealed the location of the Rap1 binding site on the wild-type Rhea F0, with V15, K17, T18, K37 and E40 being highlighted at the centre of this interaction. Structural models of Rap1:Rhea F0 binding agree with our findings, with the 5 highlighted residues seen to make close contact with the Rap1 switch I domain.\ud \ud Together this work confirms a direct interaction between Rhea and Rap1 whilst providing biochemical validation for the lethal phenotypes observed in mutant flies. It also provides further insight into new mechanisms of focal adhesion formation and integrin activation
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