Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1).

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein.

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtßγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtßγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

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.

Solution structure of the ESCRT-I complex by small-angle X-ray scattering, EPR, and FRET spectroscopy.

ESCRT-I is required for the sorting of integral membrane proteins to the lysosome, or vacuole in yeast, for cytokinesis in animal cells, and for the budding of HIV-1 from human macrophages and T lymphocytes. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the ubiquitin E2 variant and Vps28 C-terminal domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I. Here we have characterized the structure of ESCRT-I in solution by simultaneous structural refinement against small-angle X-ray scattering and double electron-electron resonance spectroscopy of spin-labeled complexes. An ensemble of at least six structures, comprising an equally populated mixture of closed and open conformations, was necessary to fit all of the data. This structural ensemble was cross-validated against single-molecule FRET spectroscopy, which suggested the presence of a continuum of open states. ESCRT-I in solution thus appears to consist of an approximately 50% population of one or a few related closed conformations, with the other 50% populating a continuum of open conformations. These conformations provide reference points for the structural pathway by which ESCRT-I induces membrane buds.

Structural modulation of phosducin by phosphorylation and 14-3-3 protein binding.

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the G(t)ßγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function.

Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

Maximum entropy analysis of analytically simulated complex fluorescence decays.

We tested a Maximum Entropy Method developed for oversampled data (SVD-MEM) on complex analytically simulated exponential decay data consisting of both noisy and noiseless multi-exponential fluorescence decay curves. We observed recovery of simulated parameters for three sets of data: a decay containing three exponential functions in both intensity and anisotropy curves, a set of intensity decays composed of 4, 5 and 6 exponential functions, and a decay characterized by a Gaussian lifetime distribution. The SVD-MEM fitting of the noiseless data returned the simulated parameters with the high accuracy. Noise added to the data affected recovery of the parameters in dependence on a data complexity. At selected realistic noise levels we obtained a good recovery of simulated parameters for all tested data sets. Decay parameters recovered from decays containing discrete lifetime components were almost independent of the value of the entropy scaling parameter γ used in the maximization procedure when it changed across the main peak of its posterior probability. A correct recovery of the Gaussian shaped lifetime distribution required selection of the γ-factor which was by several orders of magnitude larger than its most probable value to avoid a band splitting.

The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms.

Yeast 14-3-3 protein isoforms BMH1 and BMH2 possess a distinctly variant C-terminal tail which differentiates them from the isoforms of higher eukaryotes. Their C-termini are longer and contain a polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which occupies the ligand binding groove and blocks the binding of inappropriate ligands. Whether the same holds true or not for the yeast isoforms is unclear. Therefore, we investigated the conformational behavior of the C-terminal segment of BMH proteins using various biophysical techniques. Dynamic light scattering, sedimentation velocity, time-resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that the molecules of BMH proteins are significantly larger compared to the human 14-3-3zeta isoform. On the other hand, the sedimentation analysis confirmed that BMH proteins form dimers. Time-resolved tryptophan fluorescence experiments revealed no dramatic structural changes of the C-terminal segment upon the ligand binding. Taken together, the C-terminal segment of BMH proteins adopts a widely opened and extended conformation that makes difficult its folding into the ligand binding groove, thus increasing the apparent molecular size. It seems, therefore, that the C-terminal segment of BMH proteins does not function as an autoinhibitor.

14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3).

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.

Creatine kinase structural changes induced by substrates.

Myofibrillar creatine kinase (CK) buffers the cellular ATP concentration during fluctuating ATP turnover in a muscle. In order to detect structural changes of the CK molecule due to bound substrates, the dynamics of free, ATP-bound, and ATP+creatine-bound CK were examined, using steady-state and time-resolved fluorescence spectroscopy. The intrinsic tryptophan fluorescence of non-labelled CK presented the smaller fluorescence lifetime 2.38 ns and rotation correlation time 27 ns for the CK-ATP (in comparison with the times 2.72 ns and 35 ns for the free CK), and their moderate return to the longer times 2.42 ns and 29 ns for the CK-ATP+creatine complex. Three conformations for the non-labelled CK were indicated also by different quenching of fluorescence by acrylamide. Data were confirmed by anisotropy experiments with CK-(FITC labelled), providing the same substrate dependence of the rotation times (34 ns, 27 ns and returning 30 ns). The results indicate the existence of three conformations arranged according to the "energy minimizing principle" by ligated substrates. In this way the data implicate another essential component of physiological control at the subcellular level in the transition of the nonreactive CK-ATP+creatine complex to the reactive enzyme molecule.

Long-term adaptation of Bacillus subtilis 168 to extreme pH affects chemical and physical properties of the cellular membrane.

We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.

Temperature modulates binding specificity and affinity of the d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis.

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.

Frequency domain fluorometry with pulsed light-emitting diodes.

We present a simple way to extend the time resolution of a standard frequency domain (FD) fluorometer by use of pulsed light-emitting diodes (LEDs) as an excitation source. High temporal resolution of the multifrequency FD method requires the excitation light to be modulated up to the highest possible frequencies with high modulation depth. We used harmonic content of subnanosecond-pulsed LEDs for generation of modulated excitation light. By a replacement of the light source, the upper frequency limit increased to 500-600 MHz, which is almost triple the frequency limit of the standard FD fluorometer equipped with an ordinary photomultiplier tube and an electro-optical modulator. Besides the increased time resolution, this approach allowed for elimination of a light modulator with an associated synthesizer and radio frequency power amplifier that are normally required for FD measurements with continuous wave light sources. Performance of the instrument with pulsed LED excitation is demonstrated on several examples of ultraviolet-excited fluorescence decays. We show that pulsed LEDs can serve as an inexpensive alternative to pulsed laser sources for FD fluorescence spectroscopy.

Shared CaM- and S100A1-binding epitopes in the distal TRPM4 N terminus.

The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function. The TRPs tails contain a number of conserved epitopes that specifically bind the intracellular modulators. Here, we identify new binding sites for the calmodulin (CaM) and S100 calcium-binding protein A1 (S100A1), located in the very distal part of the TRPM4 N terminus. We have used chemically synthesized peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1-binding sites. We have found that the ligands binding epitopes at the TRPM4 N terminus overlap, but the interacting mechanism of both complexes is probably different. The molecular models supported by data from the fluorescence method confirmed that the complexes formations are mediated by the positively charged (R139, R140, R144) and hydrophobic (L134, L138, V143) residues present at the TRPM4 N terminus-binding epitopes. The data suggest that the molecular complexes of TRPM4/CaM and TRPM4/S100A1 would lead to the modulation of the channel functions.

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states.

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.

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.

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli.

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.

Depolarization affects the lateral microdomain structure of yeast plasma membrane.

We report the transmembrane voltage-induced lateral reorganization of highly-ordered lipid microdomains in the plasma membrane of living Saccharomyces cerevisiae. Using trans-parinaric acid (all-trans-9,11,13,15-octadecatetraenoic acid) as a probe of lipid order and different methods of membrane depolarization, we found that depolarization always invokes a significant reduction in the amount of gel-like microdomains in the membrane. Different depolarization mechanisms, including the application of ionophores, cell depolarization by an external electric field, depolarization by proton/hexose co-transport facilitated by HUP1 protein and a reduction of membrane potential caused by compromised respiration efficiency, yielded the same results independently of the yeast strain used. The data suggest that the voltage-induced reorganization of lateral membrane structure could play significant role in fast cellular response to acute stress conditions, as well as in other membrane microdomain-related regulatory mechanisms.

Short-lived fluorescence component of DPH reports on lipid--water interface of biological membranes.

Fluorescence measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) in large unilamellar phospholipid vesicles were performed to characterize the influence of the membrane physical properties on the short-lived lifetime component of the fluorescence decay. We have found that the short-lived component of DPH significantly shortens when the membrane undergoes a temperature-induced phase transition as it is known for the long-lived component of DPH. We induced membrane phase transitions also by alcohols, which are reported to be distributed different way in the membrane--ethanol close to the membrane-water interface and benzyl alcohol in the membrane core. A different effect of the respective alcohol on the short and long decay component was observed. Both the time-resolved fluorescence spectra of DPH taken during lipid vesicle staining and the lifetime dependences caused by changes of temperature and/or induced by the alcohols show that the short-lived fluorescence originates from the population of dye molecules distributed at the membrane-water interface.

A recombinant glutamine-binding protein from Escherichia coli: effect of ligand-binding on protein conformational dynamics.

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.