F3 Subdomain Research Articles

Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways.

Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro. The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2. In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive. Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.

Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.

Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5β1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)-PINCH-parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.

Effect Of Lipids On The Sequence-Specific Interactions Of Kindlin-3 and Talin-1 With The Cytosolic Tail Of The Integrin β3 Subunit

Activating the platelet integrin αIIbβ3 is an essential step for primary hemostasis. Physiologic αIIbβ3 activation occurs when platelet agonist-generated inside-out signals induce binding of the FERM domains of the cytosolic proteins talin-1 and kindlin-3 to the cytosolic tail (CT) of the β3 subunit of αIIbβ3. While talin-1 binding is thought to activate αIIbβ3 by physically causing separation of the αIIb and β3 cytosolic and transmembrane domains, αIIbβ3 activation in platelets does not occur in the absence of kindlin-3 binding to the β3 CT. Nonetheless, it is unclear whether it is necessary for talin-1 and kindlin-3 to be concurrently bound to the β3 CT in order to activate αIIbβ3, and if that is the case, whether there is a preferred order of binding and whether binding is cooperative. It is noteworthy in this regard that the sequences of the core binding motifs on the β3 CT for the talin-1 and kindlin-3 FERM domains, N744PLY747 and N756ITY759, respectively, are quite similar. To begin to address these questions, we have expressed and purified recombinant forms of the integrin-binding talin-1 head domain (THD) and full-length kindlin-3 and measured their interaction with a peptide corresponding to the β3 CT by surface plasmon resonance (SPR). For these experiments, the β3 CT was anchored to the dextran matrix of a CM5 SPR sensor chip and the equilibrium kinetics of THD and kindlin-3 binding was measured. Analysis of the THD binding data was compatible with two classes of binding sites, a high affinity site with a Kd of 155 nM and a low affinity site with a Kd of 3.5 µM. Similar analysis of kindlin-3 binding was also consistent with two classes of binding sites, a high affinity site with a Kd of 5 nM and a lower affinity site with a Kd of 2.2 µM. Next, we tested the effect of mutating the core binding motifs for the THD and kindlin-3 on the β3 CT. We found that replacing Y759 in the core kindlin-3 binding motif with Ala eliminated high affinity kindlin-3 binding, whereas replacing Y747 in the core THD binding motif with Ala eliminated low affinity kindlin-3 binding. Conversely, the Y747A replacement eliminated high affinity THD binding, while the Y759A replacement eliminated low affinity THD binding. Thus, these experiments demonstrate that the talin-1 and kindlin-3 FERM domains each recognize the general NXXY motif, but their high affinity interactions with this motif are highly sequence-specific. Previously, we found that appending the β3 CT to acidic phospholipids increased its affinity for the THD by three orders of magnitude, likely through interactions involving an extended positively-charged surface on the THD F2 and F3 subdomains. Further, kindlins contain a pleckstrin homology domain with a conserved lipid-binding loop that has been found to be essential for αIIbβ3 activation. Accordingly, we investigated the effect on THD and kindlin-3 binding of tethering the β3 CT to DOPC-coated L1 SPR chips. Unexpectedly, we found that when the β3 CT was tethered to lipid, the Kd for THD binding increased to 430 nM, comparable to the Kd we previously found using isothermal titration calorimetry for THD binding to the β3 CT appended to liposomes. We also found that kindlin-3 binding to the β3 CT tethered to lipids was unexpectedly weaker than binding in the absence of lipid, but it remained approximately 3-fold stronger than THD binding under the same conditions. Previous NMR and hydrogen-deuterium exchange studies of the β3 CT appended to liposomes have revealed that the regions of the β3 CT containing the THD and kindlin-3 binding sites consist of two dynamic amphiphilic helices that are stabilized by interacting with lipid bilayers. Thus, the results presented here suggest that the folding of the β3 CT and the interaction of the folded structure with lipids are important determinants of the strength of the interaction of the THD and kindlin-3 with the β3 CT and consequently are important factors in the regulation of αIIbβ3 activation. Disclosures:No relevant conflicts of interest to declare.

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.

Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism

AbstractKindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2+/− mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2+/− mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2+/− mice, kindlin-2+/− endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5′-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5′-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) were increased twofold to threefold on kindlin-2+/− ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2+/− mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

The Structure of the Ternary Complex of Krev Interaction Trapped 1 (KRIT1) Bound to Both the Rap1 GTPase and the Heart of Glass (HEG1) Cytoplasmic Tail

Loss of function mutation in Krev interaction trapped 1 (KRIT1) causes autosomal dominant familial cerebral cavernous malformations and disrupts cardiovascular development. The biological function of KRIT1 requires that its FERM (band 4.1, ezrin, radixin, moesin) domain physically interact with both the small GTPase Rap1 and the cytoplasmic tail of the Heart of glass (HEG1) membrane anchor. In this study, we show that the KRIT1 FERM domain can bind both Rap1 and HEG1 simultaneously, and we solved the crystal structure of the KRIT1-Rap1-HEG1 ternary complex. Rap1 binds on the surface of the F1 and F2 subdomains, in an interaction that leaves its Switch II region accessible to other potential effectors. HEG1 binds in a hydrophobic pocket at the KRIT1 F1 and F3 interface, and there is no overlap with the Rap1-binding site. Indeed, the affinity of KRIT1 or the KRIT1-Rap1 complex for HEG1 is comparable (Kd = 1.2 and 0.96 μm, respectively) showing that there is no competition between the two sites. Furthermore, analysis of this structure revealed a specific ionic interaction between the F2 lobe of KRIT1 and Rap1 that could explain the remarkable Rap1 specificity of KRIT1. This structural insight enabled design of KRIT1(K570I), a mutant that binds Rap1 with 8-fold lower affinity and exhibits increased binding to HRas. These data show that HEG1 can recruit the Rap1-KRIT complex to the plasma membrane where Rap1's Switch II region remains accessible and reveals an important determinant of KRIT1's specificity for Rap1.

Kindlin-2

Kindlin-2

Computational Studies of Talin-Mediated Integrin Activation

Integrins are heterodimeric (αβ) cell surface receptors that are involved in many essential cellular processes, such as cell migration, as well as pathological defects like thrombosis and cancer. They are activated to a high affinity state by the formation of a complex involving the β integrin cytoplasmic tail, the membrane and the head domain of talin, a large cytoplasmic protein that links integrins to actin. Here, we model the binding and the interactions of the complete talin head domain with a phospholipid bilayer, using a multiscale approach that combines coarse-grained and atomistic molecular dynamics simulations. The talin head domain has four sub-domains F0, F1, F2 and F3, with a long positively charged loop inserted in the F1 domain. The role of this F1 loop, which is missing from the crystal structure of the talin head (PDB:3IVF), is also explored. The results show that the talin head domain binds to the membrane predominantly via positively charged regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel conformation which optimizes its interactions with the membrane. Subsequently, the same multiscale approach is used to probe the formation of the talin/membrane/α/β-tail complex and how this complex promotes a rearrangement of the integrin α and β trans-membrane (TM) helices.

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 μM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

Structural basis of the junctional anchorage of the cerebral cavernous malformations complex

The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K(d) = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr(1,380)-Phe(1,381)), thus defining a new mode of FERM domain-membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM-membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.

Biophysical analysis of Kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal β-subunit NPXY motif.

Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β(1A) and β(1D) isoforms, although the interaction is stronger with β(1A). An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

Scaffold Protein Tethering Ability under Load: FAK's FERM Domain Mechanical Properties V. Binding Site

Mechanotransductive scaffolding proteins are tethered and thus subjected to mechanical loads that potentially induce partial or total unfolding. Focal adhesion kinase (FAK) is regulated by mechanical stimulation through extracellular matrix (ECM) proteins and actin cytoskeleton contractility. FAK is composed of three major domains: two of which putatively perform tethering (the FERM and FAT domains) while the central kinase domain is catalytically active in a wide variety of cell motility/invasion pathways upon activation. The so-called “basic patch” is the ligand-binding site on FERM's F2 subdomain, which is connected, via an unstructured loop, directly to FERM's F3 subdomain and distally to the kinase. As a mechanically competent tether, the FERM domain must carry loads between the basic patch and the F3 subdomain's C-terminal. A key question is whether these subdomains lose their tertiary structure under load_and therefore unwrap into a “beads on a string” configuration_and, if so, what consequences this has for ligand-binding subdomain stability. Towards an understanding of the FERM domain's ability to tether a mechanically competent FAK (pdb: 2al6), FERM's unfolding pathways are studied via Steered Molecular Dynamics (SMD). SMD simulations of the unfolding process reveal force peaks, extended conformations of intermediate states, and intra-molecular load pathways. Loads are applied to FERM's C-terminal and a set of residues in the basic patch known to bind to both phospholipids and phosphopeptides. By differentially applying loads to the basic patch's secondary structures, unfolding behavior, including both force levels and intermediate states, is revealed. Pulling-mode simulations_mimicking AFM_identify unfolding intermediate states; constant-force-mode simulations probe the structural behavior of identified intermediates. Given the diversity of ligands known to bind to the basic patch, mechanical behavior as a function of binding-site secondary structure is crucial for understanding mechanotransduction.

Capturing Spontaneous Membrane Insertion and Membrane-Induced Conformational Changes of Talin at an Atomic Resolution

Integrins are a diverse set of proteins that play a central role in complex biological processes, such as tumor metastasis and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. The cytoskeletal-associated protein talin constitutes one of the major activation pathways of integrin through a membrane-mediated mechanism. While the involvement of activated, membrane-bound talin in this process is well established, atomic details of membrane binding of talin and of talin-dependent integrin activation have been lacking. Using our novel, highly mobile membrane mimetic simulation system, we have successfully captured complete insertion of the talin head domain (THD) in a phosphatidylserine membrane in three independent unbiased simulations, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. Electrostatic potential calculations revealed THD to be highly polarized, providing a potential mechanism explaining how the protein is aligns for optimal encounter with the membrane. We also observe a large, membrane-induced interdomain conformational change (>2.5 nm), which brings the F3 subdomain into contact with the anionic membrane via residues K325, N326, and K327. This result explains how F2 and F3 subdomains can simultaneously bind the membrane, a biochemically established aspect that could not be explained by the crystal structure. Moreover, we characterize a phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.

Dynamic Characterization of the Membrane-Mediated Activation of Integrin by Talin At Atomic Reoslution

Abstract 2191Integrins are a diverse set of proteins that play a central role in many complex biological processes, such as embryonic development, tumor metastasis, and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. One of the major activation pathways that has come to the forefront of integrin research is the membrane-mediated activation of integrin by the cytoskeletal-associated protein talin. While the interaction between talin and integrin is well-established, an atomic-resolution description of the membrane-binding process of talin and of talin-dependent integrin activation has been lacking.Here, we present a study describing the membrane insertion process of the talin head domain (THD) and its subsequent interaction with the transmembrane domain of integrin αIIbβ3. Using our novel, highly mobile membrane mimetic simulation system, we simulated complete membrane insertion of THD in a phosphatidylserine (PS) membrane a total of six (6) times, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. However, we also observe a large, interdomain conformational change (> 2.5 nm), which brings the F3 subdomain into contact with the surface of the anionic membrane via residues K325, N326, and K327. Moreover, we characterize a novel, phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.Using the membrane-bound model of the THD generated in these simulations, we have also simulated the interaction between talin and the integrin β3 transmembrane domain. We were able to identify the well-known NPxY-PTB binding motif, in addition to a non-specific intermolecular hydrophobic pocket, which helps talin bind to the integrin β3 tail. Once bound to integrin β3, talin induces a bend of over 30° in the transmembrane domain at the surface of the membrane (Figure 1), which causes the burial of the conserved D723 within the lipid head-group region; this phenomenon has been observed in all cases. We believe this is a mechanism for integrin activation whereby the conserved salt bridge is mechanically broken by the THD, leading to separation of the α- and β-helices. [Display omitted] Finally, the separation of the the α- and β-tails apart wasstudied using slow pulling simulations. These studies have shown that the cytoplasmic ends of the helices interact rather loosely, while the extracellular endsare held together via tight packing of the helices. Free energy profiles of mutants in this region have shown significant decreases in stability of the “off-state”, while mutants in the cytoplasmic end do not show as significant an effect on the free energy landscape of the dimer. Disclosures:No relevant conflicts of interest to declare.

Multiscale simulations suggest a mechanism for integrin inside-out activation

Integrins are large cell-surface adhesion receptors that can be activated to a high affinity state by the formation of an intracellular complex between the integrin β-subunit tail, the membrane, and talin. The F2 and F3 subdomains of the talin head play a key role in formation of this complex. Here, activation of the integrin αIIb/β3 dimer by the talin head domain was probed using multiscale molecular dynamics simulations. A number of novel insights emerge from these studies, including (i) the importance of the integrin αIIb subunit F992 and F993 residues in stabilizing the "off" state of the αIIb/β3 dimer, (ii) a crucial role for negatively charged groups in the F2-F3/membrane interaction, (iii) binding of the talin F2-F3 domain to negatively charged lipid headgroups in the membrane induces a reorientation of the β transmembrane (TM) domain, (iv) an increase in the tilt angle of the β TM domain relative to the bilayer normal helps to destabilize the α/β TM interaction and promote a scissor-like movement of the integrin TM helices. These results, combined with various published experimental observations, suggest a model for the mechanism of inside-out activation of integrins by talin.

Crystallization and preliminary X-ray crystallographic analysis of the human kindlin-2 PH domain

Kindlins contribute to the correct assembly of integrin-containing focal adhesion sites through their direct interaction with the cytoplasmic tail of β-integrin. The FERM domain of kindlins has a unique subdomain organization: the F2 subdomain harbours a centrally located pleckstrin homology (PH) domain that is thought to be involved in the membrane targeting of kindlins. FERM domains are found in a number of cytoskeletal proteins that mediate the interaction between integrins and cytosolic proteins. In the present study, the PH domain ofhuman kindlin-2 was subcloned, solubly expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. A diffraction data set was collected at 2.8 Å resolution using synchrotron radiation on BL-4A at the Pohang Accelerator Laboratory (Pohang, Republic of Korea).

Abstract 3582: Targeting the FAK amino terminus F2 subdomain to disrupt growth factor receptor protein interactions, signaling and function

Abstract Introduction: Focal adhesion kinase (FAK), IGF-1R and the c-MET are tyrosine kinases that induce cell proliferation, invasion, metastases and survival. We have previously shown that FAK physically interacts with growth factor receptors in pancreatic cancer cells and is involved in tumor progression. Since IGF-1R and c-MET have significant structural similarity, we hypothesize that both interact with a similar conserved amino acid “patch” on FAK. We also hypothesize that this patch can be targeted with small molecules to inhibit pancreatic cancer essential signaling pathways and induce apoptosis. Methods: Based on Lipinski rules, we screened a chemical library of 240,000 compounds against a conserved patch on the F2 subdomain of FAK using in silico high throughput screening. We obtained the top scoring compounds from the National Cancer Institute Developmental Therapeutics Program and evaluated their effects on: 1) FAK protein interactions with c-MET and IGF-1R, 2) FAK, IGF-1R and c-MET kinase activity, 3)cell viability and apoptosis, and 4) growth of Panc-1 and Miapaca-2 pancreatic cancer cells in both orthotopic and direct xenograft mouse tumor models. Results: Based on co-immunoprecipitation assays, our lead compound blocked the interaction of FAK with both c-MET and IGF-1R in pancreatic cancer cells, without altering their kinase activity. Treatment with our lead compound led to decreased phosphorylation of AKT, inhibited cell viability and induced apoptosis in a dose-dependent manner (range 2-20μM). In addition, the combination of our lead compound with either 5-FU or gemcitabine chemotherapy resulted in a synergistic decrease in pancreatic cancer cell viability. More importantly, 10mg/kg of lead compound via subcutaneous injection, effectively (p<0.05) caused pancreatic tumor regression in vivo in both an orthotopic and direct xenograft model of pancreatic cancer. Conclusions: Our data suggest that targeting the protein interactions of FAK with growth factor receptors including c-MET and IGF-1R has specific therapeutic effects that can increase the sensitivity of pancreatic cancer cells to conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3582. doi:10.1158/1538-7445.AM2011-3582

Membrane Insertion and Membrane-Induced Conformational Changes of Talin F2F3 Triggering Integrin Activation

Activation of the integrin αβ heterodimer plays an important role in important biological processes such as thrombus formation and tumor metastasis. A well-known activator of intergin is the cyoskeletal-associated protein talin, whose membrane association is suggested to initiate the activation process. However, atomic details of the membrane-binding event and subsequent activation of integrin are largely unknown. We have utilized a novel membrane mimetics system developed in our laboratory, in conjunction with equilibrium molecular dynamics (MD) simulations to describe the membrane-binding event of talin F2F3 and characterize its subsequent interaction with integrin β3. In addition to characterizing the membrane orientation patch of talin F2F3, we were able to elucidate a conserved, Phe-rich hydrophobic anchor which was suggested by mutational studies, but was not evident in previous structural studies. Additionally, crystallographic studies could not explain the binding of the F3 subdomain to the membrane, which is well-characterized in mutational studies. Utilizing the highly-mobile membrane mimetics, we were able to capture large-scale conformational changes in talin F2F3 which promote the interaction of the F3 subdomain with the membrane via several residues in the β1-β2 loop. This study represents one of the first examples of membrane-induced large-scale conformational changes observed using MD simulations. Using the membrane-bound talin F2F3 subdomain, we then studied how the integrin β3 will be affected by membrane binding of talin. These simulations revealed atomistic details of the NPxY-PTB binding motif as well as formation of the membrane-proximal hydrophobic pocket. Additionally, talin F2F3 induces the bending of integrin β3, changing the angle between the cytoplasmic and transmembrane domains by approximately 50°. This buries the conserved β-Asp within the headgroup region, possibly giving a mechanism as to how the α-β salt bridge is broken and consequently how integrin is activated.

Protein-Protein and Protein-Lipid Interactions Modulate αIIbβ3 Inside-out Signaling.

Abstract 152Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cell-extracellular matrix interactions. The platelet integrin αIIbβ3 binds soluble fibrinogen following platelet activation, an event necessary for the formation of platelet aggregates. Integrins reside on plasma membranes in a highly regulated and dynamic equilibrium between inactive resting states and active ligand binding conformations. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Agonist-induced activation of αIIbβ3 in platelets (inside-out activation) requires binding of the FERM domain of the cytoskeletal protein talin to highly conserved membrane proximal and distal sequences in the β3 subunit cytoplasmic domain. FERM domain binding to the β3 cytoplasmic domain likely disrupts the αIIbβ3 TM domain-cytoplasmic domain heterodimer. As the β3 cytoplasmic domain exists in close apposition to the inner leaflet of the plasma membrane and the talin FERM domain is known to interact with the negatively-charged phospholipids that are common in eukaryotic membranes, we have investigated the effects of the membrane environment on β3 and talin structure and the talin-β;3 protein-protein interaction that regulates αIIbβ3 activation. A water soluble form of the β3 cytoplasmic domain was expressed, purified, and chemically conjugated to phospholipid bilayers, mimicking the native environment of the αIIb and β3 cytoplasmic domains as they exit the membrane. Using circular dichroism (CD) spectroscopy, the β3 cytoplasmic domain was found to have a significant increase in secondary structure and overall helicity when attached to the bilayer. Similarly, using hydrogen-deuterium exchange (HDX) mass spectrometry, we found that there is an overall decrease in amide backbone flexibility, particularly in the membrane proximal talin-interacting region. We also found significant changes in talin secondary structure in detergents by CD and by limited proteolysis mapping, supporting dramatic conformational changes in talin upon membrane binding. Using isothermal titration calorimetry (ITC) and integrin-conjugated lipid bilayers, we investigated the effects of lipid composition on talin-bilayer and talin-β3 cytoplasmic domain interactions. We found that the talin FERM domain likely has two binding sites for the membrane-conjugated β3 cytoplasmic domain, one in the canonical F3 subdomain and a second that depends on the F2 subdomain. Moreover, the talin head domain does not bind to β3 on neutral bilayers and requires a significant excess of phosphatidylserine (PS) relative to β3 to allow saturation. Interestingly phosphatidylinositol (4, 5)-bisphosphate containing bilayers have significantly higher affinity and mitigate the need for excess PS. Our results point toward a model in which β3 cytoplasmic domain structure is dependent on the lipid environment and its interaction with talin. Thus, αIIbβ3 activation is significantly regulated by lipid which may play a role in constraining αIIbβ3 activation by talin in the crowded platelet intracellular environment. Disclosures:No relevant conflicts of interest to declare.