Protein kinases have key roles in cells as they regulate diverse signal transduction pathways. Mitogen-activated protein kinase (MAPK) signaling route modulates several processes, such as cell proliferation, cell programming, metabolic changes and stress responses. Within the group of proteins participating in this pathway, the MAPK kinase (MEK1) is a dimeric, 393-residue-long, dual-specificity protein kinase that phosphorylates both tyrosine and threonine residues. In this study, we explored the conformational changes occurring during the unfolding of MEK1, by using orthogonal biophysical techniques. Intrinsic fluorescence, extrinsic 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, dynamic light scattering (DLS), and far-ultraviolet (UV) circular dichroism (CD) showed that the protein acquired a native-like conformation within a narrow pH range (8.0 to 9.0). Urea and guanidinium hydrochloride (GdmCl) denaturations followed by intrinsic and ANS fluorescence and far-UV CD, at pH 8.1, where the protein acquired a native-like conformation, showed that: (i) the apparent conformational stability of isolated MEK1 was low; and (ii) the unfolding occurred through the presence of intermediates. The presence of several unfolding intermediates was also evidenced through: (i) differential scanning calorimetry (DSC) in the absence of the ligand ATP; and (ii) unfolding simulations with the help of computational techniques based on constraint network analysis (CNA). We propose that the apparent low stability of this protein was related to its flexibility and modulates its ability to interact with diverse molecular partners.

The inhibitor of growth 4 (ING4) acts as a tumor suppressor regulating chromatin structure. Due to this nuclear function, ING4 has a nuclear localization signal (NLS), which is recognized by the cellular translocation machinery ‒ mainly formed by proteins named importins, which include the isoform importin α 3 (Imp α 3) ‒ to allow movement through the nuclear membrane. Peptidyl arginine iminohydrolases (PADIs) are enzymes involved in the posttranslational modification of arginine to citrulline. PADI4, one of the five isoforms of PADI in humans, citrullinates ING4 at the NLS region. We studied in vitro and in silico how the different degrees of citrullination affected binding of the NLS of ING4 to PADI4, Imp α 3 and its truncated species (ΔImp α 3), lacking the importin binding domain, by using several biophysical techniques and molecular simulations. To that end, we synthesized eight peptides encompassing the NLS of ING4 (residues 130 to 152), with single, double and triple citrulline replacements at Arg132, Arg142 and Arg144. The peptides were monomeric and disordered, as tested by DOSY, 1D- and 2D-¹H-NMR experiments. All the peptides were capable of binding to PADI4 with low micromolar affinities, but their affinity decreased as the fraction of citrullination increased. Moreover, all peptides could bind to both importin species with affinities in the low micromolar range, and their affinities were also dependent on the citrullination degree. The peptides targeted the canonical NLS binding site for cargo proteins of both importin species. These findings suggest that: (i) citrullination at the NLS might interfere with ING4 nuclear translocation; and (ii) successive citrullination at the NLS affect binding to PADI4.

Cancer cells must maintain molecular mechanisms relying on an energy trade-off between resistance and key functions to survive. PADI4 (peptidyl arginine deiminase 4) is an enzyme implicated in the conversion of arginine to citrulline (citrullination), that has been related to the development of several types of cancers and in numerous inflammatory routes. Salvianolic acid A (SAA) is a stilbenoid, obtained from the radix of Salvia miltiorrhiza, with several anti-cancer and anti-inflammatory features. In this work, we report and characterize the interaction between PADI4 and SAA. The binding reaction was followed by using several biophysical probes, namely fluorescence, isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR); the affinity constant was ∼600 nM (ITC). The enzyme binding to the C-terminal region of RING1B (E3 ubiquitin-protein ligase really interesting new gene 1 B), which is a substrate of PADI4, was altered in the presence of SAA, as shown by NMR. Colorimetric assays indicated that SAA was an activator of the citrullinating activity of PADI4. Moreover, the results in silico suggested that binding of SAA to the dimeric PADI4 occurred at the dimerization interface, and not at its active site, potentially stabilizing the dimeric, active form of the protein. The effect of SAA was shown to be cell-line-dependent, and its presence in glioblastoma cells hampered the binding between intact RING1B and the enzyme. Altogether, this work shows that PADI4 was capable of binding to SAA, at a novel site, and opens the venue to develop its use in modulating in vitro and in cellular assays the deimination processes.

Xylan, the principal component of hemicellulose, requires the concerted action of endo- and exo-acting xylanolytic enzymes for its complete depolymerization into fermentable sugars. This study reports the biochemical and thermodynamic characterization of a glycoside hydrolase family 10 endo-β-1,4-xylanase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (GsXynA), cloned and heterologously expressed in Escherichia coli BL21(DE3) and purified to homogeneity by immobilized metal-ion affinity chromatography. The 338-residue enzyme behaved as a monomer and showed maximal activity on xylan substrates at pH 6.5 and 75 °C, retaining over 90% residual activity between pH 5 and 9 and remaining stable up to 70 °C, after 60 min of incubation. Kinetic and thermodynamic analyses of thermal inactivation monitored by enzymatic activity measurements revealed an enthalpy-driven denaturation process, while circular dichroism indicated that this irreversible unfolding is kinetically, not thermodynamically, controlled. GsXynA hydrolyzed beechwood xylan mainly to xylobiose and xylotetraose, consistent with mixed endo and exo activity, and was uncompetitively inhibited by 4-hydroxybenzoic acid. Combined action with the β-xylosidase GsXynB2 markedly increased D-xylose release, reaching a 53.23% hydrolysis yield and a degree of synergy of 3.79 after 24 h. These results demonstrate that GsXynA is a thermostable, versatile xylanase with promising potential for lignocellulosic biomass bioconversion.