The receptor tyrosine kinase cKIT plays a pivotal role in a variety of physiological processes and is implicated in a broad spectrum of pathological conditions. Its activity is controlled by phosphorylation-dependent conformational changes, which also influence the binding mode of small-molecule inhibitors. We employed an integrated experimental and computational strategy to characterize the conformational landscape of cKIT and to elucidate the binding mechanisms of four inhibitors, Avapritinib, Olverembatinib, Labuxtinib and Cenisertib. A combination of Surface Plasmon Resonance (SPR), enzymatic assay, Molecular Dynamics (MD) simulations, stability energy evaluation, Limited Proteolysis coupled to Mass Spectrometry (LiP-MS) and X-Ray crystallography was used to investigate both active and inactive kinase states. Avapritinib preferentially binds the active form of cKIT, as supported by SPR kinetics, LiP-MS patterns, and MD results. Structural data further confirm that this compound occupies the ATP-binding pocket, consistent with a Type I inhibitor. Olverembatinib and Labuxtinib exhibit high affinity for the inactive kinase, showing stronger binding to the inactive form by SPR and inducing extensive protection of residues spanning the ATP-binding pocket in LiP-MS experiment. MD analysis reveals the burial of key pocket residues, supporting a Type II inhibition mode. Cenisertib exhibits a more complex behavior. While SPR indicates binding to both kinase states, LiP-MS, MD and X-Ray crystallography analyses reveal distinct interaction patterns depending on phosphorylation. Overall, this work highlights how distinct inhibitors exploit different conformational states of cKIT and demonstrates the value of integrating structural analyses, biophysical measurements, calculations and molecular simulations to define the mechanism of kinase inhibition.
Irene Cipollone, Carmen Gratteri, Carmine Talarico et al.· International Journal of Bio...· 0 citations
Protein-based biosurfactants remain underexplored compared to glycolipids and lipopeptides, despite their unique interfacial properties and self-assembly behavior. PAC3, a surface-active protein produced by the marine fungus Acremonium sclerotigenum, exhibits dual behavior as both a biosurfactant and bioemulsifier. For this reason, it can be seen as a high molecular weight proteinaceous compound, able to efficiently reduce surface tension. Here, we identify PAC3 as the first member of a previously unrecognized family of fungal protein biosurfactants. The complete amino acid sequence of PAC3 was determined through a combined de novo transcriptomic and mass spectrometry approach, revealing an 83-residue protein that lacks the canonical eight-cysteine motif typical of hydrophobins, the most surface-active proteins known. Sequence, phylogenetic, and structural analyses revealed a distinct fold and amphipathic architecture, with a negatively charged surface and a hydrophobic planar region, providing a molecular basis for its strong interfacial activity. The identification of homologous sequences across fungi supports the existence of a novel protein family. Notably, we show through spectroscopy and confocal microscopy that PAC3 fibrils exhibit deep-blue intrinsic fluorescence, a property recently associated with amyloid architecture. To support industrial application, we developed a simplified downstream process based on methanol/chloroform extraction, reducing costs while preserving functionality. In parallel, the use of waste frying oil enhanced fungal biomass production and supported efficient PAC3 synthesis, demonstrating a sustainable production strategy. Overall, this study introduces a new class of fungal biosurfactant proteins and provides a foundation for their biotechnological exploitation.
Rossana Pitocchi, Giulia Fichera, P. Cicatiello et al.· International Journal of Bio...· 0 citations