A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

Abstract

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) - a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic "pull-push" process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic "steric clash" model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.

Figure 1

Talin-RS has a dual inhibitory role on talin-F2F3. (A) Crystal structure of talin-F2F3/talin-RS complex. The individual subunits are labeled in green for talin-F2F3 and blue for talin-RS. Arrows indicate the two fingers in talin-F2F3 grabbing talin-RS. (B) Superimposition of talin-F2F3 bound to talin-RS with that bound to β1D CT (PDB code 3G9W) showing that the membrane proximal (MP, salmon) region, but not the membrane distal (MD, red) region in β1D CT is buried in the talin-F2F3/talin-RS interface. The arrow indicates the significant movement of K318-K324 loop in talin-F3 from the inactive state (bound to talin-RS) to active state (bound to β1D CT). (C) Close-up view of talin-F2F3/talin-RS interface shown in Figure 1A. Left panel shows the amino acid side chains involved in upper finger region (K318-K324) and right panel shows the ones in bottom finger region (D369-Y377) and the β5 and β6 regions. Interface residues from talin-F3 are colored in pink and labeled in black; interface residues from talin-RS are colored in orange and labeled in red; non-interface residues K324 and L325 in talin-F3 are colored in cyan. (D) Electrostatic surface representation of the complex viewed from the top (left panel) and from the side (right panel). The blue area indicates positive charge enriched region and red area indicates negative charge enriched region. It is clear that the extensively negatively charged surface on talin-RS would disfavor the talin association with membrane (right panel).

Figure 2

The autoinhibited talin is unfavorable to bind membrane. 2D-HSQC of 0.04 mM ¹⁵N-labeled talin-F2F3 (left) or 0.04 mM ¹⁵N-labeled talin-F2F3 plus 0.08 mM unlabeled talin-RS (right) in the absence (black) and presence (red) of 2.4 mM 4:1 POPC:POPS LUV. Significant line broadening (as reflected by the decreased peak intensity) occurred for many signals (see representative insert panel) when negatively charged vesicle was added to free talin-F2F3 (left panel). Such line broadening effect was dramatically reduced (right panel) when the same vesicles were added to talin-F2F3/talin-RS complex, indicating talin-RS effectively inhibits the access of talin-F2F3 to membrane.

Figure 3

Mapping of PIP2 and PIP3-binding sites on talin-F2F3 using the corresponding head groups IP3 and IP4, respectively. ¹H/¹⁵N chemical shift changes of 0.04 mM talin-F2F3 in the presence of (A) 0.8 mM IP3 or (B) 0.2 mM IP4. Both ligands reveal two major similarly perturbed regions with IP4 having a stronger effect likely due to additional negatively charged phosphate group. (C-E) Perturbation profiles of 0.04 mM K272A/K274A (C), 0.04 mM K322A/K324A (D), and 0.04 mM K254A/K256A (E) talin-F2F3 in the presence of 0.2 mM IP4. We note that the N-terminal region of talin-F2F3 is also somewhat perturbed, which is insensitive to these mutations and thus the perturbation is probably due to some secondary effect.

Figure 4

Membrane enriched with PIP2 favorably interacts with talin-F2F3 and promotes talin unmasking via a pull-push mechanism. (A) A structural view to show how unstimulated membrane surface would repel the latent talin (left panel), whereas PIP2-enriched membrane lipids strongly “pulls” up the talin-F2F3 and “pushes” down talin-RS, leading to the interface mismatch and unmasking of the integrin site on talin-FERM (right panel). The blue area indicates region enriched with positive charge and red area indicates region enriched with negative charge. (B) Representative HSQC region of 0.06 mM ¹³C-¹⁵N-labeled talin-RS in the absence (black) and presence of 0.05 mM talin-F2F3 (blue) or 0.05 mM talin-F2F3 and 3 mM 4:1 POPC:PIP2 LUV (red) or 0.05 mM talin-F2F3 K272A/K274A mutant and 3 mM 4:1 POPC:PIP2 LUV (green). The arrows show that the addition of PIP2 vesicle competes effectively with talin-RS to talin-F2F3, but much less effectively with the talin-F2F3 K272A/K274A mutant. (C) Comparison of the activation of integrin αIIbβ3 by vector control, full-length talin (Talin), full-length talin E1714K/E1794K/E1797K/E1798K/E1808K (Talin 5EK), full-length talin M319A+5EK (Talin M319A-5EK), and full-length talin M319A (Talin M319A). Both Talin 5EK and Talin M319A are more active than Talin through different mechanisms (see the text). As expected, the combination of M319A and 5EK is more active than the individual ones (P < 0.007 for Talin vs Talin 5EK, P < 0.001 for Talin vs Talin M319, and P < 0.002 for Talin 5EK vs Talin M319A-5EK). Note that WT talin has some basal activity as shown before, consistent with that there is an equilibrium between inactive and active talin. The data are representative of seven experiments.

Figure 5

A mechanism of spatiotemporal regulation of talin in mediating integrin activation. In unstimulated cells, talin is autoinhibited and hindered from accessing membrane via the charge-charge repulsion mechanism. Agonist stimulation promotes the co-localization of PIPKIγ with talin, thereby leading to the local enrichment of PIP2 for activating talin via the electrostatic pull-push mechanism. The electrostatic pull-push process is different from known steric occlusion by PIP2 that directly interferes with the autoinhibitory protein interfaces^,.