Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 µm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

Structural Basis for the Interaction between Pyk2-FAT Domain and Leupaxin LD Repeats.

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has four leucine-aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.

Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled.

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.

Structural basis of BAK activation in mitochondrial apoptosis initiation.

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Small molecule SJ572946 activates BAK to initiate apoptosis.

Poration of the outer mitochondrial membrane by the effector BCL-2 proteins BAK and BAX initiates apoptosis. BH3-only initiators BID and BIM trigger conformational changes in BAK and BAX transforming them from globular dormant proteins to oligomers of the apoptotic pores. Small molecules that can directly activate effectors are being sought for applications in cancer treatment. Here, we describe the small molecule SJ572946, discovered in a fragment-based screen that binds to the activation groove of BAK and selectively triggers BAK activation over that of BAX in liposome and mitochondrial permeabilization assays. SJ572946 independently kills BAK-expressing BCL2allKO HCT116 cells revealing on target cellular activity. In combination with apoptotic inducers and BH3 mimetics, SJ572946 kills experimental cancer cell lines. SJ572946 also cooperates with the endogenous BAK activator BID in activating a misfolded BAK mutant substantially impaired in activation. SJ572946 is a proof-of-concept tool for probing BAK-mediated apoptosis in preclinical cancer research.

Uncovering human mixed lineage kinase domain-like activation in necroptosis.

MLKL and its obligate upstream receptor interacting protein kinase 3 are essential components of necroptosis. It is well established that MLKL is the executioner of plasma membrane rupture in necroptosis. In healthy cells MLKL is dormant. Several dormant configurations have emerged from high-resolution structural studies revealing distinct mechanisms of MLKL autoinhibition in mammals. MLKL is activated through the concerted actions of receptor interacting protein kinase 3, which phosphorylates MLKL, and, in the case of the human pathway, inositol phosphate (IP) metabolites synthesized by the IP kinases of the IP metabolic pathway. Here, we highlight recent progress toward understanding the mechanisms of regulation of human MLKL, and survey the latest opportunities for targeting MLKL in pathophysiology.

Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites.

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6 promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP6 and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP6 production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100-600 µM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites.

Intrinsic Instability of BOK Enables Membrane Permeabilization in Apoptosis.

The effector B cell lymphoma-2 (BCL-2) protein BCL-2 ovarian killer (BOK) induces mitochondrial outer membrane permeabilization (MOMP) to initiate apoptosis upon inhibition of the proteasome. How BOK mediates MOMP is mechanistically unknown. The NMR structure of the BCL-2 core of human BOK reveals a conserved architecture with an atypical hydrophobic groove that undergoes conformational exchange. Remarkably, the BCL-2 core of BOK spontaneously associates with purified mitochondria to release cytochrome c in MOMP assays. Alanine substitution of a unique glycine in helix α1 stabilizes BOK, as shown by thermal shift and urea denaturation analyses, and significantly inhibits MOMP, liposome permeabilization, and cell death. Activated BID does not activate WT BOK or the stabilized alanine mutant to promote cell death. We propose that BOK-mediated membrane permeabilization is governed in part by its unique metastability of the hydrophobic groove and helix α1 and not through activation by BH3 ligands.

Structural insight into the mechanisms of targeting and signaling of focal adhesion kinase.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a "right-turn" elongated bundle; the globular fold is mainly maintained by hydrophobic interactions. The bound peptide further stabilizes the structure. Certain signaling events such as phosphorylation and molecule interplay may induce opening of the helix bundle. Such conformational change is proposed to precede departure of FAK from focal adhesions, which starts focal adhesion turnover.

Structural features of the focal adhesion kinase-paxillin complex give insight into the dynamics of focal adhesion assembly.

The C-terminal region of focal adhesion kinase (FAK) consists of a right-turn, elongated, four-helix bundle termed the focal adhesion targeting (FAT) domain. The structure of this domain is maintained by hydrophobic interactions, and this domain is also the proposed binding site for the focal adhesion protein paxillin. Paxillin contains five well-conserved LD motifs, which have been implicated in the binding of many focal adhesion proteins. In this study we determined that LD4 binds specifically to only a single site between the H2 and H3 helices of the FAT domain and that the C-terminal end of LD4 is oriented toward the H2-H3 loop. Comparisons of chemical-shift perturbations in NMR spectra of the FAT domain in complex with the binding region of paxillin and the FAT domain bound to both the LD2 and LD4 motifs allowed us to construct a model of FAK-paxillin binding and suggest a possible mechanism of focal adhesion disassembly.

Structural and mechanistic insights into the interaction between Pyk2 and paxillin LD motifs.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) subfamily of cytoplasmic tyrosine kinases. The C-terminal Pyk2-focal adhesion targeting (FAT) domain binds to paxillin, an adhesion molecule. Paxillin has five leucine-aspartate (LD) motifs (LD1-LD5). Here, we show that the second LD motif of paxillin, LD2, interacts with Pyk2-FAT, similar to the known Pyk2-FAT/LD4 interaction. Both LD motifs can target two ligand binding sites on Pyk2-FAT. Interestingly, they also share similar binding affinity for Pyk2-FAT with preferential association to one site relative to the other. Nevertheless, the LD2-LD4 region of paxillin (paxillin(133-290)) binds to Pyk2-FAT as a 1:1 complex. However, our data suggest that the Pyk2-FAT and paxillin complex is dynamic and it appears to be a mixture of two distinct conformations of paxillin that almost equally compete for Pyk2-FAT binding. These studies provide insight into the underlying selectivity of paxillin for Pyk2 and FAK that may influence the differing behavior of these two closely related kinases in focal adhesion sites.

Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling.

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators.

Phosphorylation of paxillin LD4 destabilizes helix formation and inhibits binding to focal adhesion kinase.

Cell migration is a dynamic process that requires the coordinated formation and disassembly of focal adhesions (FAs). Several proteins such as paxillin, focal adhesion kinase (FAK), and G protein-coupled receptor kinase-interacting protein 1 (GIT1) are known to play a regulatory role in FA disassembly and turnover. However, the mechanisms by which this occurs remain to be elucidated. Paxillin has been shown to bind the C-terminal domain of FAK in FAs, and an increasing number of studies have linked paxillin association with GIT1 during focal adhesion disassembly. It has been reported recently that phosphorylation of serine 273 in the LD4 motif of paxillin leads to an increased association with Git1 and focal adhesion turnover. In the present study, we examined the effects of phosphorylation of the LD4 peptide on its binding affinity to the C-terminal domain of FAK. We show that phosphorylation of LD4 results in a reduction of binding affinity to FAK. This reduction in binding affinity is not due to the introduction of electrostatic repulsion or steric effects but rather by a destabilization of the helical propensity of the LD4 motif. These results further our understanding of the focal adhesion turnover mechanism as well as identify a novel process by which phosphorylation can modulate intracellular signaling.

GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs.

The G protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of the p21-activated kinase (PAK) and PAK-interactive exchange factor to focal adhesions, and its activation is regulated by the interaction between its C-terminal paxillin-binding domain (PBD) and the LD motifs of paxillin. In this study, we determined the solution structure of rat GIT1 PBD by NMR spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting and vinculin tail domains. Previous studies showed that GIT1 interacts with paxillin through the LD4 motif. Here, we demonstrated that in addition to the LD4 motif, the GIT1 PBD can also bind to the paxillin LD2 motif, and both LD2 and LD4 motifs competitively target the same site on the PBD surface. We also revealed that paxillin Ser(272) phosphorylation does not influence GIT1 PBD binding in vitro. These results are in agreement with the notion that phosphorylation of paxillin Ser(272) plays an essential role in regulating focal adhesion turnover.