Effect Of Lipids On The Sequence-Specific Interactions Of Kindlin-3 and Talin-1 With The Cytosolic Tail Of The Integrin β3 Subunit

Activating the platelet integrin αIIbβ3 is an essential step for primary hemostasis. Physiologic αIIbβ3 activation occurs when platelet agonist-generated inside-out signals induce binding of the FERM domains of the cytosolic proteins talin-1 and kindlin-3 to the cytosolic tail (CT) of the β3 subunit of αIIbβ3. While talin-1 binding is thought to activate αIIbβ3 by physically causing separation of the αIIb and β3 cytosolic and transmembrane domains, αIIbβ3 activation in platelets does not occur in the absence of kindlin-3 binding to the β3 CT. Nonetheless, it is unclear whether it is necessary for talin-1 and kindlin-3 to be concurrently bound to the β3 CT in order to activate αIIbβ3, and if that is the case, whether there is a preferred order of binding and whether binding is cooperative. It is noteworthy in this regard that the sequences of the core binding motifs on the β3 CT for the talin-1 and kindlin-3 FERM domains, N 744 PLY 747 and N 756 ITY 759 , respectively, are quite similar. To begin to address these questions, we have expressed and purified recombinant forms of the integrin-binding talin-1 head domain (THD) and full-length kindlin-3 and measured their interaction with a peptide corresponding to the β3 CT by surface plasmon resonance (SPR). For these experiments, the β3 CT was anchored to the dextran matrix of a CM5 SPR sensor chip and the equilibrium kinetics of THD and kindlin-3 binding was measured. Analysis of the THD binding data was compatible with two classes of binding sites, a high affinity site with a K d of 155 nM and a low affinity site with a K d of 3.5 µM. Similar analysis of kindlin-3 binding was also consistent with two classes of binding sites, a high affinity site with a K d of 5 nM and a lower affinity site with a K d of 2.2 µM. Next, we tested the effect of mutating the core binding motifs for the THD and kindlin-3 on the β3 CT. We found that replacing Y759 in the core kindlin-3 binding motif with Ala eliminated high affinity kindlin-3 binding, whereas replacing Y747 in the core THD binding motif with Ala eliminated low affinity kindlin-3 binding. Conversely, the Y747A replacement eliminated high affinity THD binding, while the Y759A replacement eliminated low affinity THD binding. Thus, these experiments demonstrate that the talin-1 and kindlin-3 FERM domains each recognize the general NXXY motif, but their high affinity interactions with this motif are highly sequence-specific. Previously, we found that appending the β3 CT to acidic phospholipids increased its affinity for the THD by three orders of magnitude, likely through interactions involving an extended positively-charged surface on the THD F2 and F3 subdomains. Further, kindlins contain a pleckstrin homology domain with a conserved lipid-binding loop that has been found to be essential for αIIbβ3 activation. Accordingly, we investigated the effect on THD and kindlin-3 binding of tethering the β3 CT to DOPC-coated L1 SPR chips. Unexpectedly, we found that when the β3 CT was tethered to lipid, the K d for THD binding increased to 430 nM, comparable to the K d we previously found using isothermal titration calorimetry for THD binding to the β3 CT appended to liposomes. We also found that kindlin-3 binding to the β3 CT tethered to lipids was unexpectedly weaker than binding in the absence of lipid, but it remained approximately 3-fold stronger than THD binding under the same conditions. Previous NMR and hydrogen-deuterium exchange studies of the β3 CT appended to liposomes have revealed that the regions of the β3 CT containing the THD and kindlin-3 binding sites consist of two dynamic amphiphilic helices that are stabilized by interacting with lipid bilayers. Thus, the results presented here suggest that the folding of the β3 CT and the interaction of the folded structure with lipids are important determinants of the strength of the interaction of the THD and kindlin-3 with the β3 CT and consequently are important factors in the regulation of αIIbβ3 activation. Disclosures: No relevant conflicts of interest to declare.