We have determined the solution structure of the talin C-terminal actin-binding domain, a five-helix bundle that has a similar fold to the HIP1R THATCH core domain (Brett et al, 2006
). Using mutagenesis, we have defined the actin-binding surface on the bundle as a region of highly conserved residues forming a hydrophobic patch lined by basic residues. Binding to F-actin is negatively regulated by helix 1 (the USH), on the opposite face of the bundle to this site, although the physiological significance of this remains to be determined. In the full-length protein, this domain is C-terminal to a series of helical bundles that make up the talin rod, and the mechanical force exerted on talin might weaken the interaction between the USH and the core of the bundle, thereby increasing its affinity for F-actin.
An intriguing feature of proteins containing a THATCH domain is the C-terminal helix that seems to enhance actin binding by supporting dimer formation. Here, we describe for the first time the structure of one of these domains, the talin dimerisation domain, which forms an antiparallel dimer. NMR indicates that additional intramolecular interactions between the unstructured regions N- and C-terminal to the dimerisation helix may also help to stabilise the antiparallel dimer. The structure suggests that full-length talin might adopt a wide variety of conformations, including an extended tail-to-tail dimer (). This notion is supported by data showing that HIP1R is a rod-shaped dimer with globular heads at either end (Engqvist-Goldstein et al, 2001
). Talin has previously been reported to form an antiparallel dimer (Goldmann et al, 1994
), which is difficult to reconcile with our results on the C-terminal domain. Mutagenesis of the dimerisation domain clearly demonstrates the importance of this domain in supporting high-affinity actin binding. Intriguingly, most mutations in the dimerisation helix rendered the domain monomeric, and F-actin binding was markedly reduced (). However, we were able to identify two mutants (R2510A and R2513A) that retained the ability to form dimers while showing a reduction in F-actin binding, suggesting that the dimerisation domain itself might contribute to actin binding.
Electron microscopy and image analysis studies together with DSC and co-sedimentation assays provide direct evidence for binding of the dimeric C-terminal domain of talin to filamentous actin. The 3D reconstruction indicates that the dimeric talin construct binds to three actin monomers along the long-pitch helix of the filament (). This is surprising because most F-actin-binding proteins tend to bind two monomers (McGough, 1998
), often involving a prominent hydrophobic pocket in the filament primarily composed of subdomain 1 (Dominguez, 2004
) with some contributions from subdomain 2 of the long-pitch neighbour below (Volkmann et al, 2000
). In our model, the upper helical bundle of the talin dimer is indeed located close to this consensus binding site on F-actin, contacting two actin monomers along the long-pitch helix. This is consistent with the hydrophobic nature of the binding site determined by mutagenesis. The second talin helical bundle is mainly bound to the front of a single actin monomer right below these two, and the dimerisation domain is close enough to make contact with the negatively charged N-terminal region of subdomain 1 in the central actin monomer of the three.
One consequence of this mode of binding and the intrinsic symmetry of the dimer is that there must be two non-equivalent modes of actin binding. This is consistent with a study on an isolated monomeric THATCH core domain (Galkin et al, 2005
) where two different modes of binding were identified. However, in contrast to that study, we were not able to observe F-actin binding by the monomeric talin five-helix bundle (equivalent to the HIP1R THATCH core domain), indicating that both sets of contacts seen in our model are necessary to produce a stable complex. The model assumes that there are no major rearrangements between the domains in solution (as determined by SAXS) and the actin-bound form of the dimer. This assumption is fully consistent with the shape of the additional density, which can be accounted for well by docking the SAXS dimer structure into the density. A slight twist of the dimer bringing the helical bundles in a more parallel position results in an even better fit. The model for the actin-bound dimer places the residues implicated in actin binding close to the filament surface and the N termini on two opposite sides of the actin filament. However, this assignment is not unique and shows only that there is no contradiction between the dimer placement in the model and the other data.
The finding that the dimeric C-terminal actin-binding site of talin binds to a single actin filament explains why this domain fails to bundle actin filaments under the conditions used in this study. In contrast, Smith and McCann (2007)
report that this domain has bundling activity, as determined by low-speed centrifugation and by negative stain electron microscopy. All our experiments using the construct lacking the USH were carried out within 24–48 h of purification, and the integrity of the fold was confirmed by DSC. We found that the protein has a tendency to aggregate with time as detected by NMR, and this may explain the discrepancy between the two studies. It is well established that full-length talin has actin-bundling activity, as does the talin rod (Schmidt et al, 1999
). However, it is important to note that talin contains at least two other regions that bind F-actin, namely the talin FERM domain (Lee et al, 2004
) and residues 951–1327 in the centre of the talin rod (Hemmings et al, 1996
). It will be important to establish the role of each of these actin-binding sites in a cellular context. Initial studies using talin1 knockout cells have shown that the C-terminal region of talin is required to support coupling of surface-associated fibronectin to the actin cytoskeleton (Jiang et al, 2003
), but further studies are required to establish whether this was due to loss of the C-terminal actin-binding site or to the fact that the protein was monomeric. The results reported here pave the way for such studies.