Solution structure of the N-terminal F0 domain of talin1. (A) Domain structure of the talin1 head; residue numbers indicate domain boundaries. (B) Amino-acid sequence alignment and secondary structure of the F0 and F1 domains. Invariant residues are in magenta and conserved residues in yellow. Buried residues are marked ‘–' (<80% exposed) and ‘^*' (<20% exposed). Phosphorylation sites are indicated by red boxes. Loop deletion regions are shown by coloured lines under the sequence; Δ30 (blue), Δ31 (red) and Δ34 (green). (C, D) Solution structure of the F0 domain; (C) superposition of the 20 lowest energy structures. (D) Ribbon diagram of the F0 structure. (E, F) Surface representations of the talin1 F0 domain. (E) Surface representation showing the position of residues conserved among talins. The sequences used in the conservation analysis are shown in Supplementary Figure 2A, colour scheme as in (B). A conserved surface comprising helix 1 and the loop between helix 1 and strand 3 (shown in the right panel) is present in all talin isoforms. (F) Electrostatic surface of talin1 F0. Residues discussed in the text are marked.

The F1 loop is required for integrin activation by the talin head. (A) CHO cells were transfected with empty pEGFP vector or cDNAs encoding GFP-tagged talin1 head, and α5β1-integrin activation was assessed by FN9-11 binding. Activation indices of transfected cells were calculated (see Supplementary data). The results represent mean±s.e. (n⩾4). (B) Expression of recombinant GFP-tagged talin1 head constructs was checked by immunoblotting cell lysates. IB, immunoblot. (C) CHO cells stably expressing αIIbβ3-integrin were transfected with empty pEGFP vector or cDNAs encoding GFP-tagged talin1 head. Cells were harvested and αIIbβ3-integrin activation in transfected cells assessed using PAC1 antibody (see Supplementary data). The results represent mean±s.e. (n⩾4). (D) Expression of recombinant GFP-tagged talin1 head constructs was checked by immunoblotting cell lysates. Statistical significance was defined as ^**P<0.01 and ^***P<0.001. (E, F) The F1 loop is not required for talin head binding to β1A-integrin tail. (E) Binding of increasing amounts of purified GST-talin head or GST-talin head ΔF1loop to β1A tail proteins was assessed in pull-down assays followed by protein staining. Binding was quantified by scanning densitometry and the amount bound (expressed as per cent maximal binding for each protein) was plotted against the input concentration. Non-linear curve fitting was performed with a one-site-binding model. The results represent mean±s.e. GST-talin head (n⩾3; R²=0.95), GST-talin head ΔF1loop (n⩾3; R²=0.91). (F) Representative experiment of GST-talin head and GST-talin head ΔF1loop binding to β1A tail. We did not detect either GST-talin head or GST-talin head ΔF1loop binding to αIIb tails (data not shown).

Function of the F1 loop. (A) Association of F1 and its mutants with SUVs as measured by a co-sedimentation assay. Top—effect of negatively charged phospholipids on binding. Lanes 1, 2—no lipid; 3, 4—POPC; 5, 6—1:4 POPS:POPC; 7, 8—1:19 PIP2:POPC. Incubation time was 1.5 h. Supernatant (S) and (P) pellet. Bottom—effect of F1 mutations on association with 1:4 POPS:POPC SUVs. Lanes 1, 2—wild type; 3, 4—F1(Δ30); 5, 6—F1(R146E,R153E,K156E). Incubation time 1 h. (B) Charge substitution within the F1 loop impairs talin head-mediated β1A-integrin activation. CHO cells were transfected with empty pEGFP vector or cDNAs encoding WT or mutant GFP-tagged talin1 head domains, and α5β1-integrin activation was assessed by assaying FN9-11 binding. The results represent mean±s.e. (n⩾4). The constructs used were wild-type talin1 head—TalinH 1–433; TalinH(RRK/EEE)—(R146E,R153E,K156E); TalinH(TRTK/EEEE)—(T144E,R146E,T150E,K156E); TalinH(TT/EE)—(T144E,T150E) and TalinHΔF1loop (residues 139–168 deleted). (inset) Expression of recombinant talin1 head constructs was checked by immunoblotting SDS–PAGE-fractionated cell lysates. Statistical significance was defined as ^*P<0.05 and ^**P<0.01. (C) Suggested function of the F1 loop—the fly-casting model: (i) on its own, the F1 loop is predominantly unstructured with only a small population in a helical state; (ii) on contact with negatively charged phospholipids, the helical state is favoured and the loop begins to fold into a helix, resulting in cluster of positive charges on one side of the helix and (iii) shortening of the loop draws the talin F0F1 domain towards the membrane. The conserved surface on F0 (shown in yellow) is exposed and accessible for an interaction with a membrane-associated protein.

Solution structure of the talin F1 domain and the F0F1 double domain. (A) Superposition of the 20 lowest energy structures of F1. (B) Ribbon diagram of the F1 structure. (C, D) Solution structure of F0F1(Δ30) minus the F1 loop. (C) Superposition of the 20 lowest energy structures. (D) Ribbon diagram of the F0F1(Δ30) structure. (E) The interface between F0 (left) and F1 (right). (F) Small angle X-ray scattering envelope reconstruction with GASBOR, showing the best fit with the F0F1(Δ30) solution structure.

Structure of the F1 loop and its function in membrane binding. (A) Superposition of the 20 lowest energy structures calculated for the F1-loop peptide in 35% TFE. Superposition was performed separately on the C-terminal helix (residues 154–167, left) and the N-terminal helix (residues 144–152, right). The middle part of the helix is not well defined in the ensemble of the calculated structure because of its lower stability, introducing a variable kink in the peptide structure. (B) Ribbon diagram of a low energy structure of the F1-loop peptide in 35% TFE corresponding to a continuous helix. Residues experiencing resonance broadening on the addition of 1:4 POPS:POPC SUVs are highlighted in red. (C) Surface charge distribution for the structure in (B). The views differ by a 180° rotation and are optimized to show the positively charged face. (D) HN/Hα region of the 2D DQF-COSY spectra of the loop peptide alone (Free) and in the presence of 2 mM SUVs composed of POPC (PC), 1:4 POPS:POPC (PC:PS) and 1:19 PIP2:POPC (PIP2) SUVs illustrating resonance broadening on addition of negatively charged lipids. (E) Secondary structure analysis of the F1-loop (145–168) peptide by circular dichroism (CD). Left—free peptide; right—superposition of the CD spectra of the free peptide (red) and the peptide in the presence of 1:4 POPS:POPC SUVs (blue) in the 215–250 nm region. Strong contribution from the SUVs to the spectrum below 215 nm prevents the detection of the peptide signal. (F) Superposition of ¹H projections of F1 HSQC spectra illustrating the reduction in resonance intensities in the presence of POPS (red) and PIP2 (black), but not pure POPC (blue).