Structural Basis for the Autoinhibition of Talin in Regulating Integrin Activation

Abstract

Summary Activation of heterodimeric (α/β) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin β membrane-proximal cytoplasmic tails (β-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin β-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.

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

“…Talin-R 1984-2344 fragment was also found to interact with talin-F3, but at a low affinity (~20-fold weaker than the talin-RS/talin-F3 interaction) unrelated to talin autoinhibition [19]. To obtain a high-resolution view of the autoinhibited talin, we screened multiple constructs related to the talin-RS/talin-F3 complex for crystallization, among which talin-F2F3 (206-405) in complex with talin-RS was eluted by gel filtration and crystallized, allowing us to solve its structure ( Figure 1A) at 2.0 Å resolution (see structural statistics in Table 1).…”

“…Previous structural mapping experiments have shown that an interdomain complex between talin-F3 and the C-terminal rod fragment (1654-1848; talin-RS) represents the principal structural unit for talin autoinhibition [19]. Talin-R 1984-2344 fragment was also found to interact with talin-F3, but at a low affinity (~20-fold weaker than the talin-RS/talin-F3 interaction) unrelated to talin autoinhibition [19].…”

“…Talin can adopt inactive and active states during dynamic cell adhesion events, but the underlying molecular basis for this transition is elusive [2]. Previous studies suggested that the inactive talin has an autoinhibited conformation, where a key integrin-binding site on talin-F3 is self-masked by a talin-R segment (talin-RS) [19,20]. Membrane lipid phosphotidylinositol-4,5-bisphosphate (PIP2) [19,21], which is locally enriched by talin-recruited PIPKIγ kinase [22,23], was shown to activate talin [19,21] and promote integrin-mediated cell adhesion [15,[24][25][26][27][28], but the detailed structural basis of this is not clear.…”

“…Membrane lipid phosphotidylinositol-4,5-bisphosphate (PIP2) [19,21], which is locally enriched by talin-recruited PIPKIγ kinase [22,23], was shown to activate talin [19,21] and promote integrin-mediated cell adhesion [15,[24][25][26][27][28], but the detailed structural basis of this is not clear. The prevailing hypothesis is that PIP2 may sterically induce the conformational change of talin [2,15,16,19,21], a mechanism widely employed in the known PIP2-mediated activation of proteins, including FERM domain proteins [29,30].…”

Structural and mechanical functions of integrins

“…The state of adhesion complexes during migration is divided into three phases: assembly, dynamic and disassembly. The assembly phase involves an increase in the concentration of the lipid second messengers, phosphatidylinositol(4,5)P 2 (PIP 2 ) and phosphatidylinositol(4,5)P 3 (PIP 3 ) at the leading edge of the cell, in conjunction with filamentous (F-) actin protrusions and the subsequent recruitment and activation of the integrin adaptor proteins talin, vinculin and FAK or kindlin and ILK (Chen and Guan 1994;Goksoy et al 2008;Legate et al 2011). This creates a positive feedback loop and causes cytoplasmic changes, such as actin cytoskeleton reorganisation, whereby cellular morphology is adjusted to prepare for migration.…”