Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Abstract

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.cytoskeleton | plasma membrane | platelet aggregation T he integrin αIIbβ3 resides on the platelet surface in equilibrium between resting and active conformations (1-5). On circulating platelets, αIIbβ3 is constrained in its inactive conformation to prevent spontaneous platelet aggregation. Similarly, the cytoskeletal protein talin-1, whose binding to the β3 cytosolic tail (CT) stabilizes the active conformation of αIIbβ3 (6-8), is sequestered away from the β3 CT (9). Besides interacting with integrins, talin-1 interacts with negatively charged phospholipids (10-12) and phosphoinositides (13)(14)(15)(16). Stimuli generated at sites of vascular damage recruit talin-1 to the platelet plasma membrane, thereby promoting αIIbβ3 activation (17-19). Much is known about the interaction of talin-1 with integrins and lipids, but the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Elucidating these interactions is important for understanding events leading to and controlling the formation of integrin complexes and for potential pharmacologic modulation of integrin signaling.Talin-1 is a 250-kD protein containing a 45-kD N-terminal head domain attached via a flexible linker to a 200-kD C-terminal rod domain (7). Because the head domain is packed against the rod domain in its inactive state (20), talin-1 recruitment to the membrane and to integrin β CTs requires disruption of this interaction (1...

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

“…Both IP3 and IP4 are quite selective, because, under the same conditions, neither perturbed talin-F0F1 (Supplementary information, Figure S3A), which also has positively charged surface for potential interaction with membranes [16]. This specificity is consistent with the talin-F2F3 being the major fragment for the lipid binding [14]. To determine the lipid-binding sites on talin-F2F3 more precisely, we examined the IP4 binding of various talin-F2F3 mutants with mutations at sites that are positively charged and perturbed by lipid head groups, including K272A-K274A (on F2) and K322A-K324A (on F3).…”

“…Figure 1D (left panel) provides the detailed illustration of the positively charged surface in talin-F2F3, which mainly involves K268, K272, K274, R277, and K278 in talin-F2, and K322 and K324 in talin-F3. This positively charged surface appears to play a dominant role in the membrane anchoring of talin-FERM [14,15,28]. Interestingly, careful examination of the talin-F2F3/talin-RS complex structure revealed a large negatively charged surface on talin-RS, which is located at the same side as the positively charged surface of talin-F2F3 ( Figure 1D, left panel).…”

“…Moreover, the structure also reveals a previously unrecognized charge-charge repulsion mechanism via talin-RS that electrostatically inhibits the talin-FERM binding to membrane ( Figure 4A, left panel). Since the membrane-binding of talin-FERM is essential for its access to integrin β CT membrane-proximal region and for enhancing the talin-FERM/integrin interaction [13][14][15][16][17], the repulsion mechanism by talin-RS may represent an effective way to negatively regulate this targeting process, which in turn promotes the cytosolic retention of the latent talin. A recent report suggested that talin-R 482-787 fragment (talin-R1) may also interact with talin-F2F3 and inhibits the talin-FERM/membrane interaction [32].…”

“…As mentioned above, talin-FERM contains a stretch of positively charged surface that associates with membrane to promote the talin-F3/β integrin-MPCT interaction [13][14][15][16][17]. Figure 1D (left panel) provides the detailed illustration of the positively charged surface in talin-F2F3, which mainly involves K268, K272, K274, R277, and K278 in talin-F2, and K322 and K324 in talin-F3.…”

“…Structural and biochemical analyses have shown that the talin-FERM binding to integrin β CT triggers the separation of a key integrin α/β CT clasp, thus facilitating the global conformational change and activation of the receptor [7,8,13]. Stretches of positively charged surfaces on F1, F2, and F3 were also found to be crucial for this process by targeting talin-FERM to the membrane and enhancing the talin-FERM/integrin β CT interaction [13][14][15][16][17]. However, how the membrane-targeting and integrin binding of talin-FERM are spatiotemporally regulated to control the dynamics of integrin adhesion is not understood.…”