Signalling between the actin cytoskeleton and integrins is mediated by a variety of actin binding proteins, but the molecular basis of the interactions between them is only partly understood. Two recently published papers in Molecular Cell pin point the structure of specific interfaces between proteins with key roles in cell migration providing new insights into their regulation and mechanism of action.

In the first study, Hanein, Volkmann and colleagues describe in detail the interaction between actin and vinculin, a highly conserved intracellular protein that links actin filaments to integrins. Previous studies have shown that vinculin normally adopts an autoinhibited conformation and only becomes active (and able to bind actin) when it is recruited to focal adhesions. The authors employed electron microscopy, computational docking and biochemistry to obtain a model, at near atomic resolution, of the F-actin-viculin binding site. Their results show that vinculin has two distinct actin-binding sites. While the first is exposed in the autoinhibited conformation and contains multiple charged residues, the second is sterically occluded by vinculin's N-terminal domain and is hydrophobic. Interestingly, actin binding induces a conformational change of the N-terminal strap - a flexible linker that connects the vinculin head and tail domain - and C-terminal loop in the vinculin tail, which facilitates its dimerization and the formation of actin bundles. The actin-binding behaviour of a vinculin mutant that lacks the N-terminal strap further supports these findings. Finally, the authors show that vinculin's affinity for actin increases in the presence of the vinculin-binding fragment of -catenin suggesting that focal adhesion proteins contribute to the localized activation of vinculin.

The second study examines the interface between filamin A and integrin receptors. Filamins are actin cross-linking proteins that like vinculin, are involved in signal transduction from membrane receptors to the cytoskeleton. By examining the interaction of various filamin A fragments with integrin 7 tails, Calderwood and colleagues find that filamin A's immunoglobulin domain 21 (IgFLNa 21) is the major binding site for integrin 7. The crystal structure of IgFLNa 21 with an integrin 7 peptide, previously shown to mediate filamin binding, reveals that three highly conserved non-polar residues on the integrin peptide (Tyr778, Ile782 and Ile786), are important for filamin binding. Further NMR and mutagenesis experiments show that Ala2272, Ala2274 and Ile2283 on IgFLNa 21 are key for integrin binding. As both integrin 1A and 3 bind IgFLNa 21 and substitutions of interface residues affect their interaction, the 7-IgFLNa 21 complex could represent a general model for integrin-filamin interactions.

Calderwood and colleagues also find that talin, another scaffold protein connecting integrins to the actin cytoskeleton, binds to the same region as filamin on integrin tails, suggesting that these proteins may compete for integrin binding. Indeed, talin causes a dose-dependent decrease in filamin binding to recombinant 7 tails in pull-down assays, and more importantly, integrin activation, which requires talin binding to the integrin, is increased in filamin A knockdown cells. These findings indicate that competition between talin and filamin is important for the regulation of integrin activity. Phosphorylation of several residues in the filamin binding site of 7 integrin inhibits filamin binding, and suggests that kinases not only modulate filamin binding but also the competition between filamin A and talin.

These two studies illustrate the wealth of information that can be gained from the detailed examination of protein interfaces. The identification of these key residues for protein-protein interactions and steric clashes between domains, will undoubtedly be important for future mutagenic and functional studies.

Author: Monica Hoyos-Flight