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Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domain that binds to actin filaments. Here, we show thatShigellaIpaA triggers major allosteric changes in the head domain leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

The interferon-inducible protein kinase R (PKR) is a key component of host innate immunity that restricts viral replication and propagation. As one of the four eIF2α kinases that sense diverse stresses and direct the integrated stress response (ISR) crucial for cell survival and proliferation, PKR's versatile roles extend well beyond antiviral defense. Targeted by numerous host and viral regulators made of RNA and proteins, PKR is subject to multiple layers of endogenous control and external manipulation, driving its rapid evolution. These versatile regulators include not only the canonical double-stranded RNA (dsRNA) that activates the kinase activity of PKR, but also highly structured viral, host, and artificial RNAs that exert a full spectrum of effects. In this review, we discuss our deepening understanding of the allosteric mechanism that connects the regulatory and effector domains of PKR, with an emphasis on diverse structured RNA regulators in comparison to their protein counterparts. Through this analysis, we conclude that much of the mechanistic details that underlie this RNA-regulated kinase await structural and functional elucidation, upon which we can then describe a “PKR code,” a set of structural and chemical features of RNA that are both descriptive and predictive for their effects on PKR.

During Shigella cell invasion, the IpaA effector targets the focal adhesion protein vinculin through three vinculin-binding sites (VBSs). Here, we report that IpaA VBS3 also binds to talin. The 2.5 Å resolution crystal structure indicates that IpaA VBS3 forms a tightly folded α-helical bundle with talin H1-H4, contrasting with bundle unraveling upon vinculin interaction. High-affinity binding of Ipa VBS3 to talin H1-H4 requires a core of hydrophobic residues conserved in vinculin binding and a pair of electrostatic interactions accounting for talin binding specificity. IpaA VBS3 does not bind to talin H1-H5 suggesting the targeting of partially activated stretched talin but not inactive talin. Consistently, IpaA VBS3 labeled filopodial and nascent adhesions and regulated their formation through talin binding. In addition, talin-binding by IpaA VBS3 was required for bacterial capture by filopodia during Shigella invasion and for the stabilization of focal adhesions in infected cells. These findings point to the functional diversity of VBSs and a critical role for talin-binding by Shigella IpaA VBS3 and possibly talin VBSs in the regulation of adhesion structures.