Analysis of the polar residues located at the head domain of focal adhesion protein vinculin under the presence of the Shigella effector IpaA and its possible implications during in vivo mechanotransduction

Abstract

Vinculin is a protein associated to linking adhesion receptors facing the outside of cells and reinforcing them by linking its intracellular domain of those receptors or, in the case of Cell-Matrix adhesions, to bind to a first level adaptor protein such as talin. The structural organization of vinculin allows it to bind on one part to specific amphipathic motifs collectively designated as vinculin binding sites (VBS), to a set of different vinculin coactivators or actin regulators, and finally a domain responsible to constantly bind to F-actin in a catch bond manner. However, the ability of vinculin to effectively bind all of those intracellular partners, is highly dependent on its structural organization. Which is critically dependent on its ability to respond to mechanical tension on the molecule itself and not necessarily to its binding capacity to VBSs and complementary activators. This is recognized as the combinatorial model of activation. Nonetheless, Shigellas IpaA effector protein is able to mimic the conformational changes associated with the ones associated with the mechanical deformation of the molecule. This model of vinculin activation is designated as the non-combinatorial model, as the presence of a single activation-partner is enough to get the same effect. This work is devoted to dig in further to develop the previous work from this lab, as we have been able to characterize the in vitro and in vivo effects of Shigellas IpaA-Cterm region as the one responsible for both inducing conformational changes in solution, as well as the formation of super-stable adhesion, associated to maturity markers as VASP and alpha actinin. Additionally the IpaA-Cterm transfection renders those cells with the ability to maintain the adhesion structures stable and even resist the action of actomyosin relaxing molecules. Which renders them as mechanically-independent adhesions. We found that residue substitution at the surface of D1 and D2 interphase, (as well as residues maintaining the D2 domain helical bundles folded), might participate in the maintaining the structural integrity and interdomain interaction during force dependent as revealed by its ability to form protein complexes in vitro and under force-independent settings, as the morphology of cellular adhesions is altered in a way different from the previously reported targeting only the D1-D5 interaction.