Structural basis of Focal Adhesion Kinase activation on lipid membranes
2020; Springer Nature; Volume: 39; Issue: 19 Linguagem: Inglês
10.15252/embj.2020104743
ISSN1460-2075
AutoresIván Acebrón, Ricardo D. Righetto, Christina Schoenherr, Svenja de Buhr, Pilar Negrete Redondo, Jayne Culley, Carlos F. Rodríguez, Csaba Daday, Nikhil Biyani, Óscar Llorca, Adam Byron, Mohamed Chami, Frauke Gräter, Jasminka Boskovic, Margaret C. Frame, Henning Stahlberg, Daniel Lietha,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle11 August 2020free access Source DataTransparent process Structural basis of Focal Adhesion Kinase activation on lipid membranes Iván Acebrón Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Ricardo D Righetto orcid.org/0000-0003-4247-4303 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christina Schoenherr Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Svenja de Buhr Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Pilar Redondo Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Jayne Culley Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Carlos F Rodríguez orcid.org/0000-0001-9166-0132 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Csaba Daday Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nikhil Biyani Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Oscar Llorca orcid.org/0000-0001-5705-0699 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Adam Byron orcid.org/0000-0002-5939-9883 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Mohamed Chami Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Frauke Gräter Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Jasminka Boskovic Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Margaret C Frame Corresponding Author [email protected] orcid.org/0000-0001-5882-1942 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Henning Stahlberg Corresponding Author [email protected] orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Daniel Lietha Corresponding Author [email protected] orcid.org/0000-0002-6133-6486 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Centro de Investigaciones Biológicas Margarita Salas, Spanish National Research Council (CSIC), Madrid, Spain Search for more papers by this author Iván Acebrón Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Ricardo D Righetto orcid.org/0000-0003-4247-4303 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christina Schoenherr Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Svenja de Buhr Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Pilar Redondo Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Jayne Culley Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Carlos F Rodríguez orcid.org/0000-0001-9166-0132 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Csaba Daday Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nikhil Biyani Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Oscar Llorca orcid.org/0000-0001-5705-0699 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Adam Byron orcid.org/0000-0002-5939-9883 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Mohamed Chami Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Frauke Gräter Heidelberg Institute for Theoretical Studies, Heidelberg, Germany Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany Search for more papers by this author Jasminka Boskovic Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Margaret C Frame Corresponding Author [email protected] orcid.org/0000-0001-5882-1942 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Henning Stahlberg Corresponding Author [email protected] orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Daniel Lietha Corresponding Author [email protected] orcid.org/0000-0002-6133-6486 Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain Centro de Investigaciones Biológicas Margarita Salas, Spanish National Research Council (CSIC), Madrid, Spain Search for more papers by this author Author Information Iván Acebrón1,‡, Ricardo D Righetto2,‡, Christina Schoenherr3, Svenja Buhr4,5, Pilar Redondo1, Jayne Culley3, Carlos F Rodríguez1, Csaba Daday4,5, Nikhil Biyani2, Oscar Llorca1, Adam Byron3, Mohamed Chami2, Frauke Gräter4,5, Jasminka Boskovic1, Margaret C Frame *,3, Henning Stahlberg *,2 and Daniel Lietha *,1,6 1Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain 2Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland 3Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 4Heidelberg Institute for Theoretical Studies, Heidelberg, Germany 5Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany 6Centro de Investigaciones Biológicas Margarita Salas, Spanish National Research Council (CSIC), Madrid, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +(44) 131 6518510; E-mail: [email protected] *Corresponding author. Tel: +(41) 61 387 32 62; E-mail: [email protected] *Corresponding author. Tel: + (34) 911 09 8016; Email: [email protected] EMBO J (2020)39:e104743https://doi.org/10.15252/embj.2020104743 See also: KM McAndrews & R Kalluri (October 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions. Synopsis Focal adhesion kinase (FAK) is activated upon recruitment to focal adhesions via its association with the cell membrane. A cryo-EM structure of FAK bound to a PI(4,5)P2 membrane reveals structural rearrangements that lead to FAK oligomerization and activation at focal adhesions. PI(4,5)P2 binding to preformed FAK dimers results in kinase domain release from autoinhibition. FAK conformational changes allow self-assembly into membrane-bound FAK oligomers. Exposure of the FAK autophosphorylation site in its membrane-bound conformation facilitates efficient autophosphorylation in trans, leading to FAK activation. Introduction Focal adhesions are large protein complexes that assemble at the cytoplasmic side of the plasma membrane and connect the actin cytoskeleton inside the cell via integrin trans-membrane receptors to the extracellular matrix outside the cell. The complex plays an important mechanical role during mesenchymal cell migration, where contraction of actomyosin stress fibers attached to focal adhesions build up a force that is transmitted via the focal adhesion complex to the extracellular matrix to gain traction needed for cell motility (Sun et al, 2016). For this to occur in an orderly fashion, allowing for concerted focal adhesion assembly at the leading edge and disassembly at the rear (Wehrle-Haller, 2012), an extensive signaling network is associated with focal adhesions, which at the systems level is integrated with other signaling networks that control proliferation and survival (Winograd-Katz et al, 2014). To fulfill these multiple roles, focal adhesions adopt a layered architecture, where actin fibers attach to a membrane distal actin regulatory layer that is connected to integrin receptors and a membrane proximal signaling layer via a force transduction layer (Kanchanawong et al, 2010). Focal adhesion kinase (FAK) is a key component of the signaling layer and acts as a hub that integrates signaling at focal adhesions. FAK is both a scaffold protein and a non-receptor tyrosine kinase that is essential during development and plays key functions in tissue repair and wound healing (Ilic et al, 1995; Ashton et al, 2010; Ransom et al, 2018; Toro-Tapia et al, 2018). FAK is overexpressed in numerous human tumors and is in particular implicated in tumor invasion and metastasis (Provenzano & Keely, 2009; Sulzmaier et al, 2014). FAK also provides survival signals causing tumor resistance (Tavora et al, 2014; Hirata et al, 2015; Jiang et al, 2016; Diaz Osterman et al, 2019); hence, FAK has been considered an important target for anti-cancer therapies (Sulzmaier et al, 2014; Lv et al, 2018). Focal adhesion kinase shares its domain structure with the closely related proline-rich tyrosine kinase 2 (Pyk2), both contain an N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain, a central kinase domain, and a C-terminal focal adhesion targeting (FAT) domain (Fig 1A). Whereas the FAT domain is responsible for targeting FAK into focal adhesions (Arold et al, 2002; Hayashi et al, 2002; Gao et al, 2004), the FERM domain of FAK plays a central role in regulating FAK function (Cooper et al, 2003; Dunty et al, 2004) and is an interaction domain that allows FAK to integrate signals from various sources (Zaidel-Bar et al, 2007; Frame et al, 2010; Horton et al, 2015). In the autoinhibited state, the FERM domain forms intramolecular interactions with the kinase domain that occludes the active site and sequesters important regulatory phosphorylation sites (Lietha et al, 2007). In this state, FAK can form FERM-mediated dimers that are stabilized by FAT interactions with the FERM domain (Brami-Cherrier et al, 2014). Signals emanating from growth factors and integrins lead to activation of FAK (Sulzmaier et al, 2014; Walkiewicz et al, 2015), but also mechanical inputs in form of stretching forces generated in focal adhesions appear important for FAK activation (Wang et al, 2001; Torsoni et al, 2003; Seong et al, 2013; Bauer et al, 2019). Located in the membrane proximal signaling layer in focal adhesions, FAK directly interacts with the membrane lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) (Cai et al, 2008; Goni et al, 2014). PI(4,5)P2 levels are upregulated in focal adhesions by the PI(4)P-5-kinase type Iγ (Di Paolo et al, 2002; Ling et al, 2002), and we previously reported that interactions with PI(4,5)P2 guide FAK through a multistep activation sequence (Goni et al, 2014). This involves PI(4,5)P2 binding via a basic patch on the FERM domain, which induces FAK oligomerization and conformational changes that trigger autophosphorylation of Y397 in the linker between the FERM and the kinase domains. Subsequently, the Src kinase is recruited via its SH2 and SH3 domains that interact with the autophosphorylated linker in FAK and Src then phosphorylates residues Y576 and Y577 in the activation loop of the FAK kinase to induce full catalytic activity (Calalb et al, 1995; Lietha et al, 2007). Although previous work defined the main steps required for integrin-mediated FAK activation (Goni et al, 2014), it did not provide a structural understanding of events occurring on the membrane. Figure 1. Cryo-EM structure of the FERM-kinase region of FAK on a PI(4,5)P2 membrane Domain structure of FAK. Important phosphorylation sites are indicated, and the FAK region used in the cryo-EM study is boxed with a dashed line. Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5× zoom of the main image. Local resolution map of the refined Apo and AMP-PNP particle, as calculated with Blocres. 14 FAK molecules, each containing a FERM and kinase domain, are fitted into the EM maps obtained from the AMP-PNP dataset. Each molecule is in a different color. Middle: Membrane distal view; right: Membrane proximal view; left: zoomed view of central symmetric dimer from the membrane distal view with locations of FERM (F) and kinase (K) domains indicated. The equivalent figure for the Apo structure is shown in Fig EV1D. Linear oligomeric assembly of FAK formed by three symmetric FAK dimers in the AMP-PNP particle shown from the membrane distal view. FERM (F) and Kinase (K) domains are labeled. FERM and kinase of the same molecule are in the same color with the kinases shaded in a lighter tone. Model of the FAK oligomeric assembly placed on a lipid bilayer. The membrane is placed by considering the KAKTLRK basic sequence in the FERM F2 lobe as main membrane-binding site (Cai et al, 2008; Goni et al, 2014). Download figure Download PowerPoint Here, we employ cryo-electron microscopy (cryo-EM) of FAK bound to a PI(4,5)P2 membrane to provide structural details of membrane-induced conformational changes and FAK oligomerization. We observe that the oligomeric FAK assembly is formed via a tightly packed symmetric FAK dimer and these dimers are linked into an oligomer via FERM–FERM interactions that have previously been observed for FAK in solution (Brami-Cherrier et al, 2014). Our structure suggests a membrane-induced release of autoinhibition by binding of FERM-mediated FAK dimers to PI(4,5)P2 via the basic patch in the FERM domain. Intriguingly, the released kinase domain reorients via a large conformational change placing the active site toward the membrane. As a result, the linker is exposed to promote efficient autophosphorylation. We show that interfaces observed in the cryo-EM structure are important for cellular functions of FAK, including cell proliferation in 3D and cancer cell invasion. Results Cryo-EM and overall architecture of FAK bound to a PI(4,5)P2 membrane For structural studies of membrane-bound FAK, we assembled purified FAK containing the FERM and kinase domains (FAK31-686) on a lipid monolayer containing PI(4,5)P2 and analyzed samples by cryo-EM. We showed previously that C-terminal regions required for FAT do not affect PI(4,5)P2 binding, oligomerization, or PI(4,5)P2-induced autophosphorylation (Goni et al, 2014). After sample optimization, we obtained regular 2D crystal-like assemblies of FAK (Fig 1B). We prepared samples in the absence (Apo) or presence of the ATP analog AMP-PNP, the latter in order to mimic an ATP-bound state and rigidify relative movements between the N-terminal (N) and C-terminal (C) lobes of the kinase domain. We collected cryo-EM datasets at different tilt angles for the Apo (0–60°) and AMP-PNP bound state (0–50°). Crystallographic data processing using 2dx (Gipson et al, 2007) resulted in low-resolution 3D maps with a resolution of ~ 20 Å (Fig EV1A). To improve the resolution, we next followed a single-particle analysis procedure by extraction of particles from 2D crystal images (Righetto et al, 2019). A summary of cryo-EM data collection and image processing parameters is shown in Table EV1. The particle exhibits a twofold symmetry and resulting 3D maps have an overall resolution of 6.32 Å (Apo) or 5.96 Å (AMP-PNP; Fig EV1B), with local maximal resolution reaching ~ 4 Å (Fig 1C). We note that 2D crystals from the AMP-PNP dataset could be classified into two different crystal packings as apparent from 2D classifications (Fig EV1C). Atomic models were generated by flexible fitting of the crystal structures of the FERM (PDB: 2AEH) and kinase domains of FAK (PDB:1MP8) into the cryo-EM maps followed by manual adjustments (Figs 1D and EV1D, Table EV2). Secondary structure elements are well defined, but side chains rarely visible with a few exceptions (Fig EV1E). An important consideration during model building was the position and connectivity of the linker residues connecting the FERM and kinase domains (residues 362–411). For this, density of a twofold symmetric FAK dimer (hereafter referred to as symmetric dimer) at the center of the AMP-PNP containing particle (zoomed in Fig 1D) was inspected, since maps are best defined in this region. Residues 362–394 are likely disordered and are not included in the model. Residues 395–401 (including the Y397 autophosphorylation site) are modeled in the apo and AMP-PNP structures bound to the FERM F1 lobe, analogous to the position seen in autoinhibited FAK and FERM-linker crystal structures (Ceccarelli et al, 2006; Lietha et al, 2007), based on density present at this position (Fig EV1F). Residues 402–411, leading to the kinase domain, are only modeled in the AMP-PNP structure based on existing density, which is however not continuous (Fig EV1G). Although we consider less likely, we cannot exclude the possibility that residues 402–411 are disordered and connect to the other kinase domain in the dimer. Importantly, the main conclusions we draw apply with either connectivity. The final Apo and AMP-PNP models are similar with an RMSD of 1.85 Å comparing FAK monomers (superimposing 593 Cα atoms) or 2.23 Å superimposing the symmetric FAK dimer (1,186 Cα atoms). Below, we describe the FAK-AMP-PNP structure unless stated otherwise. Click here to expand this figure. Figure EV1. Expanded view on cryo-EM structure EM maps obtained by crystallographic processing of images using 2dx (Gipson et al, 2007). Shown are four unit cells (dashed lines), each with parameters α = 110 Å, β = 88 Å and γ = 107° and p2 symmetry. Plot of the Fourier shell correlation over resolution. To avoid artificially inflating the FSC, the two half-maps were reconstructed from particles randomly split on a per-crystal basis. The inset shows a masked projection of the AMP-PNP map. 2D averages of particles extracted from 2D crystal images. Note that the AMP-PNP containing particles exhibit two different crystal packings. 14 FAK molecules, each containing a FERM and kinase domain are fitted into the EM maps obtained from Apo-FAK data. Each molecule is in a different color. Middle: Membrane distal view; right: Membrane proximal view; left: zoomed view of central symmetric dimer from the membrane distal view with locations of FERM (F) and kinase (K) domains indicated. The equivalent figure for the AMP-PNP structure is shown in Fig 1D. View of the FERM F1 lobe in the central region in the AMP-PNP structure that exhibits density for side chains. Cryo-EM maps indicate that the linker region (light green) containing the Y397 autophosphorylation site is bound to the FERM F1 lobe as seen in the FERM-linker and autoinhibited FERM-kinase structures (Ceccarelli et al, 2006; Lietha et al, 2007). Cryo-EM maps show partial density for the linker region 402–412 (light green and light blue) connecting the F1 bound linker with the kinase (K) domain. The view is along a twofold symmetry axis; hence, two identical regions in two FAK molecules (green and blue) are seen. In both molecules, the ribbon of the N-terminal residue in the kinase domain (T412) is colored in red. Download figure Download PowerPoint The final structure contains 14 FAK molecules, each of them assembles into closely associated symmetric FAK dimers (Fig 1D). The symmetric dimers connect via FERM–FERM contacts to neighboring dimers to form a linear oligomeric assembly (Fig 1E). Lateral contacts to this linear oligomer are likely introduced crystallographically since they differ in the two crystal forms seen in the AMP-PNP dataset (Fig EV1C), whereas the linear arrangement shown in Fig 1E is present in all crystals (AMP-PNP and Apo). To model the FAK structure on the lipid membrane (which is for the most part not visible in 3D averaged maps), we considered the reported K216AKTLRK motif on the FERM domain as primary PI(4,5)P2-binding site (Cai et al, 2008; Goni et al, 2014). As seen in Fig EV2A, this sequence aligns in all 14 FAK molecules in one plane allowing confident positioning of the lipid membrane relative to the FAK oligomer (Fig 1F). In further support of membrane placement, 3D maps show unmodeled density at the KAKTLRK motif, as well as at sites where the kinase domain contacts the modeled membrane (Fig EV2B), likely due to less mobile lipid molecules interacting with FAK at these sites. Click here to expand this figure. Figure EV2. Known PI(4,5)P2-binding sites in FAK support membrane placement The KAKTLRK sequences (colored red) known to interact with PI(4,5)P2 lipid membranes (Cai et al, 2008; Goni et al, 2014) align in all FERM domains of the cryo-EM particle in one plane allowing positioning of the membrane as shown in Fig 1F. Unmodeled density at the KAKTLRK sites (red) in FERM domains (light arrowheads) and unmodeled density at K621/K627 residues (cyan) in the kinase domains (dark arrowheads) coincide with the position of the modeled membrane and likely represent lipid molecules with restricted mobility due to their interactions with FAK. Download figure Download PowerPoint FAK oligomerizes on the membrane via multiple interfaces The resulting structural assembly reveals several new interfaces as well as interactions observed previously. Within the symmetric FAK dimer, new interactions are formed intramolecularly between the FERM and kinase domains (F–K and F’-K’ in Fig 2A) and intermolecularly between FERM and kinase domains (F–K’ and F’-K) as well as between two kinase domains (K–K’). Bridging dimers into an oligomeric assembly occurs via FERM–FERM interactions (F–F* and F′–F** in Fig 2A) in a manner described previously to occur for FAK in solution (Brami-Cherrier et al, 2014). This interface involves W266 and is seen in 3D crystal structures that contain the FAK FERM domain. Superposition of this crystallographic FERM dimer with FERM domains in our cryo-EM structure shows a highly similar FERM–FERM interaction (Fig 2B). For newly observed interactions within the symmetric dimer, EM maps provide limited details since sidechains are mostly not defined. We therefore performed Molecular Dynamics (MD) simulations on the symmetric FAK dimer to orient sidechains for nearby interactions, while the FAK backbone was restrained by EM maps. Analysis of the interactions throughout the simulations with PISA (Krissinel & Henrick, 2007) and SC (Lawrence & Colman, 1993) reveals rather extensive interactions of predominantly polar (intramolecular F–K), mixed (intermolecular F–K′), or hydrophobic (intermolecular K–K’) character and significant shape complementarity (Table 1; Fig 3A–C), with highest energy gain suggested to result from the K–K’ interaction. In addition to protein–protein interfaces, we also observe contacts with the modeled membrane, other than the KAKTLRK sequence in the FERM domain used for membrane positioning. The kinase domain is positioned lying flat on the membrane with main contacts made via the basic residues K621 and K627 in the kinase C-lobe (Fig 3D). In this conformation, the active site of the kinase domain is facing toward the membrane. Inspection of the electrostatic potential of the membrane-interacting surface of the symmetric FAK dimer reveals in addition to the KAKTLRK basic patch a large basic region on the kinase domain that includes K621 and K627 (Fig 3E). In agreement with our structural findings, these residues have been reported previously to contribute to PI(4,5)P2 binding in FAK (Hall & Schaller, 2017). Figure 2. Interfaces in the FAK oligomeric assembly on the membrane Close-up of the central symmetric dimer of the AMP-PNP particle from the membrane distal view. Interfaces within the symmetric dimer are formed intramolecularly between the FERM and kinase domains (F and K; F’ and K’). Intermolecular interfaces are formed within the symmetric dimer between F and K’, F’ and K as well as K and K’. Intermolecular interactions that link symmetric dimers are formed between two FERM domains (F and F*; F’ and F**). Coloring is as in Fig 1E. The FERM subdomains F1 and F3 (F2 is not visible from this view), kinase N- and C-lobes (KN, KC), AMP-PNP and the N- and C-termini (N, C) are labeled. Superposition of W266-mediated FERM–FERM 3D-crystallographic dimers (Ceccarelli et al, 2006) (yellow; PDB: 2AEH) and the FERM–FERM contacts seen in the cryo-EM structure to link symmetric dimers (F–F*; F’–F**). The view is 90° rotated from panel (A). Subdomains and termini are labeled, and disordered regions in the linker are shown as dashed line. Download figure Download PowerPoint Table 1. FAK interfaces Interface Area (Å2) ΔGhydrophobicaa Calculated by PISA (Krissinel & Henrick, 2007). (kcal/mol) ΔGpolaraa Calculated by PISA (Krissinel & Henrick, 2007). (kcal/mol) ΔGtotalaa Calculated by PISA (Krissinel & Henrick, 2007). (kcal/mol) SCbb Shape complementarity, calculated by SC (Lawrence & Colman, 1993). F–K 431 ± 73 3.2 ± 2.2 −8.3 ± 2.3 −5.1 ± 1.5 0.54 ± 0.08 F–K’ 640 ± 109 −3.8 ± 1.9 −5.5 ± 2.5 −9.3 ± 3.3 0.52 ± 0.10 K–K’ 623 ± 61 −9.5 ± 2.0 −3.4 ± 1.7 −12.9 ± 1.7 0.51 ± 0.07 Interfaces: F–K: intramolecular FERM–kinase; F–K′: intermolecular FERM–kinase; K-K′: intermolecular kinase–kinase. a Calculated by PISA (Krissinel & Henrick, 2007). b Shape complementarity, calculated by SC (Lawrence & Colman, 1993). Figure 3. Key interactions at interfaces A–C. Details of intramolecular F–K (A) and intermolecular F–K’ (B) and K–K’ (C) interactions as seen in simulations restrained by EM maps. Side chains of main interacting residues are shown in stick representation, and hydrogen bonds are shown as orange dashed lines. D. The kinase is oriented with the ATP-binding site facing the membrane. Key interactions between the kinase and the membrane are made by K621 and K627. The side chains of K621, K627, and K216 (the latter is within the KAKTLRK motif of a neighboring FER
Referência(s)