Fig 1.
Domain organization of talin and kindlin and primary structures of the F1 loops.
The FERM domain of talin, also called talin head, and kindlin is sub-divided into several subdomains F0, F1, F2 and F3. The FERM domain of kindlin also contains a PH domain inserted within the F2 subdomain. The full-length talin also contains a rod domain which is absent in kindlins. The F1 domains of talin and kindlin contain a loop insert for membrane interactions. The primary structures of F1 loop of kindlin-1, kindlin-2 and kindlin-3 and talin are shown.
Fig 2.
NMR characterization and secondary structures of the F1 loop of kindlin-3.
(panel A) Two-dimensional 15N-1H HSQC spectrum of 83-residue long F1 loop of kindlin-3 showing resonance assignments of 15N and amide proton single bind correlations. The F1 loop of kindlin-3 contains residues L142-Q224, but numbered here as L1-Q83. HSQC cross-peaks arising from the additional N-terminal tag residues are marked as ‘t’. (panel B) Secondary chemical shift of 13Cα chemical shift for individual residues of F1 loop of kindlin-3.
Fig 3.
Lipid specific interactions of SUVs with the F1 loop of kindlin-3.
Overlay of 15N-1H HSQC spectra of F1 loop at different concentrations, 0 mM (black contour), 4 mM (red contour) and 6 mM, of POPC (panel A), POPS (panel B) and POPG (panel C) SUVs. Bar diagrams showing attenuations of 15N-1H HSQC cross-peak intensity of F1 loop of kindlin-3 after additions of 2 mM (black bar) and 4 mM (white bar) of POPC (panel D), POPS (panel E) and POPG (panel F) SUVs, respectively.
Fig 4.
Secondary conformations and micelle interactions of the F1 loop of kindlin-3.
(panel A) Far UV-CD spectra of F1 loop of kindlin-3 in aqueous buffer solution (in solid black line), in solutions containing 50 mM DPC (in black squares) and 50 mM SDS (in white squares). (panel B) Overlay of 15N-1H HSQC spectra of the F1 loop of kinldin-3 in aqueous buffer solution (black contour), in solutions containing either in 50 mM (red contour) or 100 mM (green contour) DPC. (panel C) Overlay of 15N-1H HSQC spectra of the F1 loop of kinldin-3 in aqueous buffer solution (black contour), in solutions containing either in 50 mM (red contour) or 100 mM (green contour) SDS.
Fig 5.
Conformational characteristics of EK21 peptide fragment derived from N-terminus of the F1 loop.
(panel A) Far UV CD spectra of EK21 peptide in free buffer solution (solid line) and in solutions containing either 50 mM DPC (solid line with black squares) or 50 mM SDS (solid line with white squares). (penal B) Bar diagram showing secondary chemical shifts of αH resonances of residues of EK21 in solutions containing SDS micelles. (panel C) Selected section of 1H-1H two-dimensional NOESY spectrum of EK21 peptide obtained in aqueous solutions containing 200 mM perdeuterated SDS.
Fig 6.
Localization of the FERM domain of kindlin in membrane.
A hypothetical cartoon showing proximity of the FERM domain of kindlin to the lipid membrane and interactions with the beta cytosolic tail. The FERM domain interacts with the phospholipid lipid bilayer (shown as black stick with round head) through F0 subdomain (in blue), PH (in purple) and potentially with split F2 (in green) and F3 (in pink) subdomains by ionic interactions with exposed positively charged surface. The unfolded F1 loop of the F1 subdomain (in red) might insert into the lipid membrane using N-terminal positive charged residues of poly-lys and sidechain of hydrophobic residues. Such a membrane localization of the FERM domain of kindlin might bring the F3 subdomain for optimal interactions with the beta cytosolic tail attached with its TM (in light purple) for activation of integrins.