Dynamic Characterization of the Membrane-Mediated Activation of Integrin by Talin At Atomic Reoslution

Abstract 2191

Integrins are a diverse set of proteins that play a central role in many complex biological processes, such as embryonic development, tumor metastasis, and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. One of the major activation pathways that has come to the forefront of integrin research is the membrane-mediated activation of integrin by the cytoskeletal-associated protein talin. While the interaction between talin and integrin is well-established, an atomic-resolution description of the membrane-binding process of talin and of talin-dependent integrin activation has been lacking.

Here, we present a study describing the membrane insertion process of the talin head domain (THD) and its subsequent interaction with the transmembrane domain of integrin αIIbβ3. Using our novel, highly mobile membrane mimetic simulation system, we simulated complete membrane insertion of THD in a phosphatidylserine (PS) membrane a total of six (6) times, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. However, we also observe a large, interdomain conformational change (> 2.5 nm), which brings the F3 subdomain into contact with the surface of the anionic membrane via residues K325, N326, and K327. Moreover, we characterize a novel, phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.

Using the membrane-bound model of the THD generated in these simulations, we have also simulated the interaction between talin and the integrin β3 transmembrane domain. We were able to identify the well-known NPxY-PTB binding motif, in addition to a non-specific intermolecular hydrophobic pocket, which helps talin bind to the integrin β3 tail. Once bound to integrin β3, talin induces a bend of over 30° in the transmembrane domain at the surface of the membrane (Figure 1), which causes the burial of the conserved D723 within the lipid head-group region; this phenomenon has been observed in all cases. We believe this is a mechanism for integrin activation whereby the conserved salt bridge is mechanically broken by the THD, leading to separation of the α- and β-helices.

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Finally, the separation of the the α- and β-tails apart wasstudied using slow pulling simulations. These studies have shown that the cytoplasmic ends of the helices interact rather loosely, while the extracellular endsare held together via tight packing of the helices. Free energy profiles of mutants in this region have shown significant decreases in stability of the “off-state”, while mutants in the cytoplasmic end do not show as significant an effect on the free energy landscape of the dimer.