Seiffert, P. | Search bioRxiv

bioRxiv2022

The prolactin receptor scaffolds Janus kinase 2 via co-structure formation with phosphoinositide-4,5-bisphosphate

Class 1 cytokine receptors transmit signals through the membrane by a single transmembrane helix to an intrinsically disordered cytoplasmic domain that lacks kinase activity. While specific binding to phosphoinositides has been reported for the prolactin receptor (PRLR), the role of lipids in PRLR signalling is unclear. Using an integrative approach combining NMR spectroscopy, cellular signalling experiments, computational modelling and simulation, we demonstrate co-structure formation of the disordered intracellular domain of the human PRLR, the membrane constituent phosphoinositide-4,5-bisphosphate (PI(4,5)P2) and the FERM-SH2 domain of the Janus kinase 2 (JAK2). We find that the complex leads to accumulation of PI(4,5)P2 at the transmembrane helix interface and that mutation of residues identified to interact specifically with PI(4,5)P2 negatively affects PRLR-mediated activation of signal transducer and activator of transcription 5 (STAT5). Facilitated by co-structure formation, the membrane-proximal disordered region arranges into an extended structure. We suggest that the co-structure formed between PRLR, JAK2 and PI(4,5)P2 locks the juxtamembrane disordered domain of the PRLR in an extended structure, enabling signal relay from the extracellular to the intracellular domain upon ligand binding. We find that the co-structure exists in different states which we speculate could be relevant for turning signalling on and off. Similar co-structures may be relevant for other non-receptor tyrosine kinases and their receptors.

biophysics↗

bioRxiv2022

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins

Diffusion measurements by pulsed field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle X-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modelling and for force field benchmarking and parameterization. SIGNIFICANCEAccurate models of the conformational properties of intrinsically disordered proteins rely on our ability to interpret experimental data that reports on the conformational ensembles of these proteins in solution. Methods to calculate experimental observables from conformational ensembles are central to link experiments and computation, for example in integrative modelling or the assessment of molecular force fields. Benchmarking such methods is, however, difficult for disordered proteins because it is difficult to construct accurate ensembles without using the data. We here circumvent this problem by combining independent measures of protein compaction to test several methods to calculate the hydrodynamic radius of a disordered protein, as measured by pulsed field gradient NMR diffusion experiments, and find the Kirkwood-Riseman model to be most accurate.

biophysics↗