The redox-active site of thioredoxin is directly involved in apoptosis signal-regulating kinase 1 binding that is modulated by oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase 5, which mediates various stress signals including oxidative stress. The catalytic activity of ASK1 is tightly controlled by oligomerization and binding of several cofactors. Among these cofactors, thioredoxin stands out as the most important ASK1 inhibitor, but only the reduced form of thioredoxin inhibits ASK1 by direct binding to its N-terminal domain. In addition, oxidation-driven thioredoxin dissociation is the key event in oxidative stress-mediated ASK1 activation. However, the structural mechanism of ASK1 regulation by thioredoxin remains unknown. Here, we report the characterization of the ASK1 domain responsible for thioredoxin binding and its complex using NMR spectroscopy and chemical cross-linking, thus providing the molecular basis for ASK1: thioredoxin complex dissociation under oxidative stress conditions. Our data reveal that the N-terminal domain of ASK1 adopts a fold resembling the thioredoxin structure while retaining substantial conformational plasticity at the thioredoxin-binding interface. Although oxidative stress induces relatively moderate structural changes in thioredoxin, the formation of intramolecular disulfide bridges leads to a considerable conformational rearrangement of the thioredoxin-binding interface on ASK1. Moreover, the cysteine residue at position 250 of ASK1 is the key element of this molecular switch. Finally, our results show that the redox-active site of thioredoxin is directly involved in ASK1 binding that is modulated by oxidative stress, thereby identifying a key target for the structure-based drug design.

Modulating FOXO3 transcriptional activity by small, DBD-binding molecules.

FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.

Forkhead Domains of FOXO Transcription Factors Differ in both Overall Conformation and Dynamics.

FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.

14-3-3 protein masks the nuclear localization sequence of caspase-2.

Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).

CaMKK2 kinase domain interacts with the autoinhibitory region through the N-terminal lobe including the RP insert.

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca⁠2+/calmodulin-dependent kinase (CaMK) family, functions as an upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase. Thus, CaMKK2 is involved in the regulation of several key physiological and pathophysiological processes. Previous studies have suggested that Ca2+/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear. METHODS: In this study, hydrogen-deuterium exchange coupled to mass spectrometry, time-resolved fluorescence spectroscopy, small-angle x-ray scattering and chemical cross-linking were used to characterize Ca2+/CaM binding-induced structural changes in CaMKK2. RESULTS: Our data suggest that: (i) the CaMKK2 kinase domain interacts with the autoinhibitory region (AID) through the N-terminal lobe of the kinase domain including the RP insert, a segment important for targeting downstream substrate kinases; (ii) Ca2+/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca2+/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca2+/CaM transiently interacts with the CaMKK2 kinase domain. CONCLUSIONS: Interactions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca2+/CaM binding the autoinhibitory region inhibits CaMKK2 by both blocking access to the RP insert and by affecting the structure of the ATP-binding pocket. GENERAL SIGNIFICANCE: Our results corroborate the hypothesis that Ca2+/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs.

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2).

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca2+/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown. METHODS: The interaction between phosphorylated CaMKK2 and the 14-3-3γ protein, as well as the architecture of their complex, were studied using enzyme activity measurements, small-angle x-ray scattering (SAXS), time-resolved fluorescence spectroscopy and protein crystallography. RESULTS: Our data suggest that the 14-3-3 protein binding does not inhibit the catalytic activity of phosphorylated CaMKK2 but rather slows down its dephosphorylation. Structural analysis indicated that the complex is flexible and that CaMKK2 is located outside the phosphopeptide-binding central channel of the 14-3-3γ dimer. Furthermore, 14-3-3γ appears to interact with and affect the structure of several regions of CaMKK2 outside the 14-3-3 binding motifs. In addition, the structural basis of interactions between 14-3-3 and the 14-3-3 binding motifs of CaMKK2 were elucidated by determining the crystal structures of phosphopeptides containing these motifs bound to 14-3-3. CONCLUSIONS: 14-3-3γ protein directly interacts with the kinase domain of CaMKK2 and the region containing the inhibitory phosphorylation site Thr145 within the N-terminal extension. GENERAL SIGNIFICANCE: Our results suggested that CaMKK isoforms differ in their 14-3-3-mediated regulations and that the interaction between 14-3-3 protein and the N-terminal 14-3-3-binding motif of CaMKK2 might be stabilized by small-molecule compounds.

Cysteine residues mediate high-affinity binding of thioredoxin to ASK1.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.

Biophysical and structural characterization of the thioredoxin-binding domain of protein kinase ASK1 and its interaction with reduced thioredoxin.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.