Biology subjects

Plaza-Menacho, I.

Publications and source records attributed to Plaza-Menacho, I..

3 recordsLinked to original sources

bioRxiv2025

The oncogenic CCDC6-RET fusion product is a dual ATP and ADP-dependent kinase that functions via cis-phosphorylation

Gene fusions products involving protein kinases are known drivers in human cancers and actionable targets for personalized therapy, yet the structural and molecular determinants that control their function are largely unexplored. Here we show that a CCDC6-RET fusion product, a driver and therapeutic target in lung and thyroid cancers, is a highly active dimeric kinase in solution. Time-resolved mass spectrometry analysis together with a robust biochemical and biophysical characterization reveal that CCDC6-RET functions as a dual ATP- and ADP-dependent kinase able to bind both nucleotides and uses them as phosphoryl donors. We also identify a crosstalk between the c-terminal and the activation segments, uncovering a mutually exclusive dependency by the former on activation loop phospho-sites controlling both the processing and the catalytic activity of the fusion protein. Furthermore, we generated a 3D-assembly of a CCDC6-RET homodimer combining electron microscopy (EM) single particle, small-angle X-ray scattering (SAXS) and in silico structural analyses. Our structural model together with cross-linking mass spectrometry data demonstrated that CCDC6-RET forms a face-to-face trans-inhibited dimer in the apo state characterized by intermolecular-crosslinked activation segments. Upon nucleotide binding and catalytic domains reorientation, fast activation loop phosphorylation is driven by a mechanism in cis. Our work uncover for the first time the molecular and structural determinants that controls CCDC6-RET function and provides a solid framework to study the structure and function of other RET fusion products.

biochemistry↗

bioRxiv2022

An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

Auto-phosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlaying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src auto-phosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular phosphorylation in which a c-terminal palindromic phospho-motif flanking Tyr 530 on the susbtrate molecule engages the P-loop of the active kinase for ready entry prior catalysis. Perturbation of the phospho-motif accounts for c-Src disfunction as indicated by viral and a colorectal cancer (CRC) associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 and global auto-phosphorylation, and this detrimental effect is caused by the susbtrate molecule inhibiting allosterically the active kinase. Our work reveals a bi-directional crosstalk between the activation and c-terminal segments that controls the allosteric interplay between susbtrate and enzyme acting kinases during auto-phosphorylation HighlightsO_LIA bi-directional phospho-switch connecting the activation and c-terminal segments controls c-Src function C_LIO_LIActivation-loop Tyr 419 is required for c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and non-catalytic function as a substrate C_LIO_LIBiochemical and structural visualization of c-Src Tyr 530 intermolecular auto-phosphorylation C_LIO_LIA double-phosphorylated c-Src on both the activation and c-terminal segments is a fully active protein C_LIO_LICancer associated c-terminal deleted variants inhibit allosterically c-Src activity by a dominant negative effect C_LI

biochemistry↗

bioRxiv2019

Hybrid histidine kinase activation by cyclic di-GMP- mediated domain liberation

Cytosolic hybrid histidine kinases (HHKs) constitute major signalling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses and kinetic modelling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudo-receiver domain Rec1 thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically control activity. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC=\"FIGDIR/small/675454v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (11K):\norg.highwire.dtl.DTLVardef@8304borg.highwire.dtl.DTLVardef@19a9d61org.highwire.dtl.DTLVardef@1d27000org.highwire.dtl.DTLVardef@b74d75_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗