Crystal Structure of the FERM Domain of Focal Adhesion Kinase*

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

The adhesion of cells to the extracellular matrix is mediated by the integrin family of cell surface receptors (1). Extracellular matrix engagement triggers conformational changes and clustering of integrins leading to the remodeling of protein complexes at focal adhesions (2). These intracellular complexes contain actin-cytoskeletal proteins and signaling molecules that regulate a number of processes including cell migration, proliferation, and differentiation (3). Focal adhesion kinase (FAK) 3 is present at focal adhesions and becomes both tyrosine-phosphorylated and activated in response to integrin signaling (4 -6). As a potential mediator of integrin signaling, it is important to understand how FAK is activated and regulated.
FAK and the related tyrosine kinase Pyk2 (also referred to as CAK-␤, RAFTK, or CADTK) comprise a subfamily of non-receptor proteintyrosine kinases defined by a central tyrosine kinase domain flanked by an amino-terminal FERM domain and a carboxyl-terminal domain. The latter domain contains a proline-rich region that interacts with other signaling molecules and a distal focal adhesion targeting domain (Fig.  1A). Autophosphorylation of FAK at Tyr 397 within a linker region between the FERM and kinase domain creates a binding site for SH2containing proteins such as Src family kinases, the p85 subunit of phosphatidylinositol 3-kinase, and Grb7 (7)(8)(9)(10)(11). An adjacent RXXPXXP motif serves as a binding site for the Src family SH3 domain and contributes to the efficient recruitment of Src family kinases to FAK (12,13). Subsequent phosphorylation of tyrosines (Tyr 576 and Tyr 577 ) within the kinase domain of FAK by the associated Src family kinase leads to the formation of an active signaling complex (14). Through phosphorylation of additional proteins assembled at the cytoplasmic tails of integrins, FAK and Src promote signaling events that modulate cellular growth, survival, and migration (6,15,16).
The amino terminus of FAK and Pyk2 share a region of homology with membrane-cytoskeletal linker proteins ezrin, radixin, and moesin (referred to collectively as ERM proteins) (17). The band 4.1 and ERM homology domain (FERM domain) is a module of ϳ300 amino acids found in a number of proteins that are membrane targeted (18). FERM domains of ERM proteins interact directly with the cytoplasmic regions of transmembrane receptors (19 -21). In addition, association of the FERM domain with the phosphoinositide phosphatidylinositol 4,5-P 2 has been demonstrated both structurally and with vesicle co-sedimentation (22)(23)(24). Mutagenesis of the basic residues within the FERM domain that mediate the phosphoinositide interaction alters the subcellular distribution of the protein (24,25). These findings suggest that both protein-protein and protein-lipid interactions may be responsible for the efficient localization of FERM-containing proteins to membrane targets.
In addition to a general role as a membrane localization module, the FERM domain may serve additional regulatory functions. Among the 518 protein kinases annotated within the human genome, FERM domains are not a common accessory domain and are only found within FAK and Janus kinase families (26). The association of Janus kinases with the cytoplasmic tails of cytokine receptors is dependent on the FERM domain (27)(28)(29)(30). Mutations in the Janus kinase 3 FERM domain inhibit receptor binding and may abrogate kinase activity (31).
To date, several interactions involving the FERM domain of FAK have been described including those with the cytoplasmic tails of inte-grins, the pleckstrin homology domain of the Tec-family kinase Etk, and the FERM domain of ezrin (32)(33)(34)(35). Association with activated growth factor receptors and the promotion of cell migration in response to growth factor signaling is dependent on an intact FERM domain of FAK (36). Immunolocalization experiments have suggested that the FERM domain of FAK is sufficient for localization to membrane substructures such as at the leading edge of lamellipodia and cell-cell junctions, and although not directly responsible for focal adhesion targeting, can modulate the localization of the protein (37,38). Finally, a direct interaction between the FERM and kinase domains of FAK may regulate the activity of the kinase (39 -42). Interestingly, sequences in the F2 subdomain of the FAK FERM domain play a role in activation of FAK signaling following cell adhesion (40). The physical nature of these interactions remains to be elucidated.
To better understand the function of the FERM domain of FAK, and as a starting point for dissecting its inter-and intramolecular interactions, we have determined its structure in two crystal forms at resolutions of 2.5 Å and 2.35 Å. The tri-lobed architecture of ERM family FERM domains is preserved in FAK (22,(43)(44)(45)(46)(47), but a unique orientation of the F3 subdomain alters the global conformation of the domain as compared with all previously studied FERM domains. The structures show that the phosphatidylinositol 4,5-P 2 binding site found in ERM family FERM domains is not preserved in FAK. Additionally, the structures reveal that the FERM-kinase linker segment masks a potential protein interaction site on the F3 lobe; the corresponding site in the talin and radixin FERM domains has been shown to bind the cytoplasmic tails of ␤ integrins and ICAM-2, respectively (21,48). We hypothesize that this masking interaction could be released by binding of Src family proteins to their adjacent docking sites in the linker.

EXPERIMENTAL PROCEDURES
Materials-Restriction and modifying enzymes were purchased from New England Biolabs. Oligonucleotides were from Sigma; B834 Escherichia coli cells were from Novagen. Seleno-L-methionine was from Sigma; metal chelating, ion exchange, and gel filtration columns were from Amersham Biosciences.
Protein Expression and Purification-The FERM domain of avian FAK was amplified by PCR from cDNA (Swiss-Prot accession number Q00944). DNA fragments encoding amino acids 1-405, 31-405, and 31-399 were each ligated into a modified pET vector containing an amino-terminal His 6 tag with a tobacco etch virus cleavage site and confirmed by DNA sequencing. The expression of recombinant protein in E. coli BL21(DE3) cells was induced at an optical density (A 600 ) of 0.8 by the addition of 0.2 mM isopropyl ␤-D-thiogalactopyranoside and grown for an additional 12-16 h at 18°C. The cells were harvested and the pellets were stored frozen at Ϫ70°C. Cell pellets were thawed in buffer A (20 mM Tris, pH 8.0, 200 mM NaCl, 5 mM imidazole, 5 mM ␤-mercaptoethanol) with the addition of 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 1 mM benzamadine, 10 mg/ml lysozyme, and 1 mg/ml DNase. Cell membranes were disrupted by sonication on ice and supernatants obtained following a 30-min centrifugation at 50,000 ϫ g were loaded onto a Ni-chelating column. Protein was eluted with buffer A containing 200 mM imidazole. FERM domain-containing fractions were pooled and the His 6 tag was cleaved by incubation with 1 g of tobacco etch virus protease per mg of protein overnight at 4°C. The concentration of NaCl was reduced to 50 mM by dilution, and the protein was loaded onto a HiTrap Q column and eluted with buffer A containing 500 mM NaCl. The FERM domain was concentrated and loaded onto a Superdex 75 size exclusion column previously equilibrated in buffer A containing 100 mM NaCl. The eluted FERM domain of FAK was greater than 95% pure as estimated by SDS-PAGE and was concentrated to 6 -10 mg/ml and stored at Ϫ70°C.
Selenomethionine (SeMet)-substituted protein was prepared using the methionine auxotroph B834(DE3) strain. Cells were grown in M9 minimal media supplemented with SeMet (40 g/ml). The subsequent steps for SeMet-labeled FERM domain proceeded as described for the native protein.
Structure Determination-The program SOLVE was used to determine the position of 10 selenium atoms from two copies of the FERM domain in the asymmetric unit in the FAK 399 structure. The initial electron density map was improved by solvent flattening and phaseextension to 2.5-Å resolution using program DM (49). A partial model was built automatically using the program ARP/wARP (50). The structure was completed and refined with several rounds of manual rebuilding using the program O and atomic refinement with CNS. The model was assessed using PROCHECK (51) and waters were added by CNS. The refined model contains residues 33-363 of FAK, whereas residues 31-32 and 364 -399 were not visible in the electron density maps and presumed to be disordered.
The structure of FAK 405 was phased by molecular replacement using the previously determined FAK 399 FERM structure as the search model and the program Phaser (52). Difference electron density maps revealed that the ordered portion of one of the two molecules in the asymmetric unit extended to residue 375, and also revealed density corresponding to residues 394 -403 and 394 -401 in the linker regions of the two molecules. These regions were built and the FAK 405 structure was refined as described above for the FAK 399 structure.

Domain Mapping, Crystallization, and Structure Determination-
The amino-terminal fragment of FAK (residues 1-405) containing the predicted FERM domain and autophosphorylation site Tyr 397 was expressed in E. coli and purified. Limited proteolysis with the protease trypsin and amino-terminal sequencing of the resulting polypeptide identified a cleavage event that removed the NH 2 -terminal 28 residues of FAK (data not shown). Analysis of the FERM region of FAK and PYK2 from a number of species revealed that sequence conservation initiated with Arg 35 (numbering refers to avian FAK). A truncated protein containing residues 31-399 (referred to here as FAK 399 ) was generated and produced monoclinic crystals that diffracted x-rays to better than 2.5-Å resolution. The FAK 399 structure was determined by multiwavelength anomalous diffraction methods using SeMet-substituted protein. We also obtained crystals with a longer construct spanning residues 31-405 (FAK 405 ); this structure was determined by molecular replacement using the FAK 399 structure as a model. The two structures are essentially the same; the only notable exception is that in the FAK 405 structure, we observe electron density for a portion of the FERM-kinase linker corresponding to the Src SH3 binding site (residues 364 to 375) and for the segment containing the Tyr 397 autophosphorylation site (residues 394 -403). These segments are not ordered or completely present in the FAK 399 crystals. Our description of the structure below is based upon the FAK 399 crystals, but we refer explicitly to FAK 399 or FAK 405 as necessary for correctness and clarity.
The FAK 399 protein crystallized in monoclinic space group P2 1 with two molecules in the asymmetric unit. The final model contains two molecules of the FERM domain and 243 water molecules and has been refined to an R value of 19.6% (R free ϭ 24.9%) at 2.5-Å resolution with excellent stereochemistry ( Table 1). The first 2 residues (Gly 31 -Ala 32 ) and the COOH-terminal 37 residues (Lys 364 -Glu 399 ) in each molecule are not visible in the electron density; this disordered region includes the Src SH3 binding site (Arg 368 -Pro 374 ) and the Tyr 397 phosphorylation site. FAK 405 protein crystallized in the orthorhombic space group P2 1 2 1 2 1 , also with two molecules in the asymmetric unit. Residues Ser 307 -Arg 312 , Leu 376 -Glu 393 , and Glu 404 -Asp 405 were not observed in the electron density and are not included in the final model. The FAK 405 structure has been refined to an R value of 21.0% (R free ϭ 25.2%) with good stereochemistry (Table 1 and "Experimental Procedures").
Structure of the FAK FERM Domain-The FAK structure reveals the three-lobed architecture characteristic of the archetypal FERM domains of ERM family members ezrin (47), radixin (22), and moesin (43) (Fig. 1B). The three subdomains (labeled F1, F2, and F3) intimately associate with one another to form a compact structure with the overall shape of a cloverleaf. Each of the three lobes of the FERM domain bears striking similarity to otherwise unrelated single-domain protein structures as previously noted (43). The F1 lobe spans residues 33-127 and exhibits a ubiquitin-like fold (53) consisting of a five-stranded ␤-sheet capped by an ␣-helix. The F2 subdomain (residues 128 -253) is all ␣-helical and contains a core 4-helix bundle similar to that found in the acyl-CoA-binding protein (54). The F3 lobe (residues 254 -352) is a ␤-sandwich capped by a COOH-terminal ␣-helix that very closely resembles a pleckstrin homology domain. The pleckstrin homology domain shares the same three-dimensional fold with other modular signaling domains including phosphotyrosine binding and EVH1 domains in addition to the F3 lobe of the FERM domain (55). It has evolved distinct binding sites for diverse protein and phospholipid ligands (56). Both phosphotyrosine binding domains and the F3 lobe of some FERM domains bind peptide ligands in a groove formed between one edge of the ␤-sandwich and the COOH-terminal ␣-helix. Interestingly, this groove is occupied in the FAK FERM domain by residues just COOH-terminal to the FERM domain in the FERM-kinase linker segment (Fig. 4). This interaction is observed in both FAK 399 and FAK 405 and is discussed more fully in a following section.
The two protein molecules in the asymmetric unit are quite similar. They superimpose with a root mean square (r.m.s.) deviation of 0.9 Å over 330 amino acids. The only significant structural deviations in the two molecules are found in loop regions (the loop connecting ␣2 and ␣3 in the F2 lobe, the loop between strands ␤3 and ␤4, and the loop between strands ␤5 and ␤6 loop in the F3 lobe). Similarly, the FAK 405 structure superimposes well with that of FAK 399 (r.m.s. deviation of 1.2 Å for 324 equivalent residues). Both structures reveal a similar noncrystallographic dimer interface, but the contact between the two molecules is small (it buries ϳ400 Å 2 of surface area on each monomer) and is therefore not suggestive of a biologically relevant dimer. Also, the isolated FERM domain elutes in size-exclusion chromatography at a volume consistent with a monomeric protein (data not shown). However, FAK self-association is thought to play a role in its catalytic regulation and we cannot exclude the possibility that the FERM-FERM interaction observed in the crystal lattice is part of a larger interaction in the intact kinase.
Comparison with the Radixin FERM-The FAK FERM domain is rather distantly related to other FERM domains. For example, it shares only 12-15% sequence identity with ERM family members (Fig. 1C). This considerable sequence divergence is reflected in significant structural changes in the FAK FERM domain as compared with other FERM domain structures studied to date. As expected, the overall fold of the FERM domain is preserved, but marked differences are observed both in the structures of the individual lobes and in their relative orientations within the domain as a whole (Fig. 2). In contrast, the FERM domains of merlin and ERM family members much more closely resemble one another (they superimpose with overall root mean square deviations on the order of 1 Å or less). We compare the FAK FERM domain with that of radixin, a well studied ERM family member. The 64-residue core of the F1 subdomain superimposes with that of radixin with an r.m.s. deviation of 1.4 Å (Fig. 2A). The second helix in the F1 subdomain (F1-␣2) is approximately two turns longer in FAK than in ERM family members. Additionally, the ␤1-␤2 and ␣2-␤5 loop regions are longer, and contain 3 10 helices not found in other FERM structures. These insertions account for the 12 additional residues within the F1 domain of FAK as compared with the structurally aligned FERM domain of radixin (Fig. 1C). Structural similarity with the F2 subdomain of radixin is limited to helices ␣1 to ␣4; the 78 residues in this 4-helix core superimpose with an r.m.s. deviation of 1.6 Å. The FAK F2 lobe includes four structural elements that are not seen in other FERM structures; these include three segments of 3 10 helix and a 9-residue helix ␣2Ј, all of which are inserted between core helices ␣2 and ␣3 (Figs. 1C and  2A). The F3 lobes of FAK and radixin superimpose with an r.m.s. deviation of 1.5 Å over 73 residues for 6 of the 7 ␤-strands and the ␣1 helix. The relatively modest structural differences between the F3 subdomains of FAK and radixin include a shift in the position of the ␤3-␤4 loop, a 3 10 helix within ␤4-␤5 turn in FAK, and a 7-residue insertion in the loop connecting strands ␤5 and ␤6.
Perhaps the most striking structural difference between the FAK FERM domain and other intact FERM structures determined to date is the relative orientation of the subdomains. In particular, the position of the F3 lobe with respect to the F1 and F2 lobes is markedly different in FAK. The FAK FERM domain is shown superimposed with the structurally related radixin and merlin FERM domains in Fig. 2B. The FAK F3 subdomain is rotated and translated such that its ␣1 helix is positioned 3.5-4 Å closer to the F1 lobe and its ␤5 strand is ϳ9 Å closer to the F1 lobe than the equivalent regions in radixin and merlin. This difference is Secondary structure elements are labeled as shown in the sequence alignment below. C, structure-based sequence alignment of FERM domains with conserved residues highlighted in yellow and identical residues in red. Avian FAK (cFAK), human FAK (hFAK), human Pyk2 (hPYK2), human Ezrin (hEz), human radixin (hRad), human moesin (hMoe), human merlin (hMer), and human band 4.1R (h41R) are shown. much greater than the shift described in comparison of the active and inactive-tail bound structures of moesin and ezrin (47,57). The net effect of this rearrangement is a significant difference in the interfaces of F3 with both F1 and F2 as compared with other intact FERM structures. The F3 lobe is less intimately associated with the F2 lobe in FAK (a buried surface area of 749 Å 2 as compared with 990 Å 2 of contact in radixin). In contrast, the F1-F3 interface is more intimate (compare 1207 Å 2 buried in FAK with 1004 Å 2 in radixin). Interacting residues between the F1 and F3 domains of FAK include His 99 with Glu 338 , Arg 127 with Asp 342 , and Asn 339 with Val 95 and Trp 97 . This novel positioning of F3 against F1 is the same in the two independent FAK molecules in the crystallographic asymmetric unit and also observed in the FAK 405 structure, which has different crystal packing contacts.
The divergent structure of the FAK FERM domain also indicates a divergence in function. The FERM domains of ERM proteins bind phosphoinositide phosphatidylinositol 4,5-P 2 ; this interaction is important for localization of the ERM proteins in cells (24). The radixin FERM domain has been crystallized in complex with inositol 1,4,5-trisphosphate (the soluble head group of phosphatidylinositol 4,5-P 2 ) (22). The IP 3 head group binds radixin in a shallow, basic cleft between the F1 and F3 domains (Fig. 3B). The FAK FERM domain clearly cannot bind phosphoinositides in the analogous position (Fig. 3, A and C). In FAK, the F1-F3 cleft is different in both structure and amino acid composition. The interface between the F1 and F3 domains of FAK is narrow relative to other FERM structures because of the divergent position of the F3 lobe as described above. Additionally, the presence of acidic residues and the lack of basic residues within this cleft is expected to preclude phosphoinositide coordination at this site in the FERM domain of FAK (Fig. 3, C and D).
The FERM-Kinase Linker Segment-In human FAK, the FERM domain ends at residue 352, and the kinase domain begins at approximately residue 415 (58). The intervening 60-residue linker segment is important for regulation of FAK function, as it contains the Tyr 397 phosphorylation site (which when phosphorylated is bound by the Src SH2 domain) and also the binding site for the Src SH3 domain. The linker is largely disordered in FAK 399 , however, in both the FAK 399 and FAK 405 structures the first 10 residues of the linker bind in the cleft between the F1 and F3 lobes and in the putative peptide recognition groove on the F3 lobe (Fig. 4). In the longer FAK 405 structure, the ordered region extends to Lys 375 and encompasses the Src SH3 docking site. Additionally, a discontinuous segment of clear density is observed for residues 394 -   FERM domain. A, the F1, F2, and F3 subdomains of avian FAK (shown in cyan) and human radixin (shown in yellow) were superimposed individually. The subdomains are highly related but notable structural differences are described in the text. B, superposition of the intact FERM domains of FAK (cyan), radixin (yellow), and merlin (red). The different orientation of FAK F3 with respect to the F1 and F2 lobes is apparent in comparison with the overall structural conservation between radixin and merlin characteristics of all ERM family and closely related FERM domains studied to date.
Just COOH-terminal to the ␣1 helix of F3, the polypeptide chain turns back into the F1-F3 cleft, and makes numerous interactions with both the F1 lobe and the F3 binding groove (Fig. 4, A and C). Contacts with the F1 lobe include a salt-bridge hydrogen bond network involving Arg 361 in the linker and the side chains of Asp 101 and His 89 in the F1 lobe, and also a hydrogen bond between the carbonyl of Thr 355 and the side chain of Arg 65 in the F1 lobe. The linker interaction with the F3 binding groove is stabilized by hydrophobic interactions of Phe 358 , Ile 359 , and Ile 360 and through a parallel ␤-sheet interaction of residues Phe 358 to Ile 360 with the ␤5 strand in the F3 binding groove (Fig. 4C). In the FAK 399 structure, the linker region beyond Pro 362 no longer contacts the FERM domain. However, in one of the two molecules in the FAK 405 structure, the ordered region extends through the Src SH3 binding site (residues 368 -375), which interacts with the surface of the F3 lobe (Fig.  5, A and B). The RALPSIP sequence (the key residues of the RXXPXXP Src SH3 binding motif are underlined) adopts a polyproline type II helical conformation and packs against the exposed ␤-sheet of the F3 lobe. Contacts with the F3 subdomain include 5 backbone-mediated hydrogen bond interactions including two with the side chains of Gln 303 and Gln 317 , and hydrophobic interactions involving Leu 370 and Ile 373 (Fig.  5B). Interestingly, polyproline motifs bind to EVH1 domains of Ena/ Vasp proteins, which also share the pleckstrin homology domain fold of the F3 lobe, in a topologically similar location (59). The orientation and specific interactions of the proline motif in the present structure, however, is not the same as that observed in EVH1 complexes.
The segment containing the phosphorylation site Tyr 397 packs against the F1 subdomain of both FERM molecules (Fig. 5, A and C).
This polypeptide consists of residues 394 -403 and forms a 3 10 helix and short ␤-strand that extends the anti-parallel ␤-sheet formed by the ␤1 and ␤2 strands of F1. This interface buries a total of 1047 Å 2 and comprises 7 backbone-mediated hydrogen bonds with only Thr 62 of F1 making a specific side chain interaction and two side chain-mediated interactions of Glu 403 with Ser 54 and His 41 (Fig. 5C). There are no previously identified examples of this surface of a FERM domain making a proteinprotein interaction. The side chain of Tyr 397 is surface exposed and in close proximity to Lys 70 . The last ordered residue of the COOH-terminal segment, Glu 403 , is near Lys 38 , His 41 , His 75 , and the cleft region between the F1 and F2 subunits. Note that it is not possible to ascertain whether the interaction of this segment is intramolecular or whether it occurs between adjacent molecules in the crystal lattice because we do not observe density for the preceding 20 residues. Either connectivity is crystallographically plausible, but the fact that this construct is monomeric in solution suggests that the interaction is intramolecular.

DISCUSSION
The F3 binding groove, occupied by the NH 2 -terminal portion of the linker segment in the present structure, is a well characterized interaction site on FERM domains. In ERM family proteins, this groove is occupied by part of the COOH-terminal actin-binding tail, which makes intramolecular interactions with the FERM domain (43). Upon activation, this autoinhibitory conformation is thought to be released, allowing interaction with the cytoplasmic tails of cognate cell-surface receptors (60). Indeed, the tail region of ICAM-2 was recently demonstrated to bind at this site in a crystal structure of radixin complexed with an ICAM-2 peptide (21). The peptide forms a ␤-sheet interaction with the ␤5 strand of the F3 lobe, thereby extending the ␤-sheet (Fig. 4B). Additionally, the corresponding site in the talin FERM domain was observed to bind an NPXY motif in the ␤3 integrin tail (48). Interestingly, this binding site is also analogous to that bound by NPXY motif sequences on the structurally homologous phosphotyrosine binding domain, as seen, for example, in the insulin receptor substrate-1 phosphotyrosine binding domain (61). Although the linker segment in FAK binds to the F3 lobe in the same location, its mode of binding is quite different from that in the structures noted above. The linker segment binds in the opposite orientation, thus forming a parallel rather than anti-parallel ␤-sheet interaction with strand ␤5 (Fig. 4).
The significance of the interaction of the NH 2 -terminal portion of the linker with the F3 lobe is unclear. It is possible that this portion of the linker segment is simply an extension of the fold of the FAK FERM domain itself, and therefore represents a structural divergence between FAK and other FERM domains. Alternatively, it is tempting to speculate that it represents a regulatory interaction, masking all or part of the binding site of an interacting protein. Like talin, FAK may associate with ␤-integrin tails. If it is a regulatory interaction, the structure suggests that it might be released by binding of a Src family protein to the SH3 and/or SH2 binding sites. Indeed, binding of a Src SH3 domain to the RALPSIP sequence would preclude its interaction with the surface of the F3 lobe and might also destabilize the interaction of the linker in the F3 groove by steric effects. The phosphorylation of Tyr 397 is a key regulatory event in the activation of FAK and scaffolding properties that recruit SH2-domain containing proteins such as Src family kinases, phosphatidylinositol 3Ј-kinases, and Grb family adaptor proteins (62). Phosphorylation and subsequent binding to the Tyr 397 site will also require its dissociation from the surface of the FERM domain.
Although structurally unrelated, the interactions of the linker segment with the FERM domain are reminiscent of the intramolecular interactions between the SH3 domain and the SH2 kinase linker segment in Src family kinases. It is tempting to speculate that one or both of the linker contacts may additionally participate in autoinhibitory interactions with the kinase, in analogy with Src kinases where the SH3 and linker together bind the N-lobe of the kinase (63). In FAK, association of SH2-containing proteins may be an important step in the activation of the kinase by facilitating disassembly of the FERM-linker and or FERM/ kinase intramolecular interactions. In loose analogy with Src kinases, phosphorylation of Tyr 397 and subsequent recruitment of a Src family kinase to the linker segment might be expected to stabilize an open and active conformation of FAK. The present structure will facilitate mutagenesis studies designed to test these possibilities and to further dissect the intramolecular interaction of the FERM domain with the kinase domain. The Src SH3 binding site is shown in green (residues 363-375) and the Tyr 397 -containing segment (residues 394 -403) is colored in pink. B, detail of the interactions between the Src SH3-binding site and the surface of F3. Hydrogen bonds between the RXXPXXP motif (residues Arg 368 to Pro 374 ) and the F3 lobe are indicated by dashed lines and involve the side chains of Gln 303 and Gln 317 on the F3 lobe. C, details of the interaction between the FAK autophosphorylation Tyr 397 segment and the F1 lobe. Hydrogen bonds are indicated with dashed lines and involve the side chains of Glu 403 with His 41 and Ser 54 bonds. Tyr 397 is surface exposed and not phosphorylated in the structure.