Koss, H. | Search bioRxiv

bioRxiv2020

Cadherin-11 dimerization multi-site kinetics: combined partial unfolding and strand-swapping

Cadherin extracellular domain 1 (EC1) mediates homophilic dimerization in adherens junctions. Conserved Trp2 and Trp4 residues in type II cadherins anchor the EC1 A-strand intermolecularly in strand-swapped dimers. Herein, NMR spectroscopy is used to elucidate the roles of Trp2 and Trp4 in Cadherin-11 dimerization. The monomeric state, with the A-strand and Trp side chains packed intramolecularly, is in equilibrium with sparsely populated partially and fully A-strand-exposed states, in which Trp2 (and Trp4, respectively) side-chain packing is disrupted. Exchange kinetics between the major state and the partially (fully) A-strand-exposed state is slow-intermediate (intermediate-fast). A separate very fast process exchanges ordered and random-coil BC loop conformations with populations dependent on A-strand exposure and dimerization status. Additionally, very slow processes connect the folded A-strand-exposed conformation to partially unfolded states, which may represent additional domain-swapping intermediates. The dimerization mechanism of type II cadherins is revealed as coupled folding and strand-swapping.

biophysics↗

bioRxiv2019

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

Receptor tyrosine kinases (RTKs), the largest class of transmembrane cell surface receptors, initiate signalling pathways which regulate diverse cellular processes. On activation these receptors rapidly recruit multiple downstream effector proteins to moderate affinity tyrosyl phosphate (pY) binding sites. However, the mechanism for expedient downstream effector protein recruitment via random molecular diffusion through the cytoplasm is not fully understood. One way in which the probabilistic outcome associated with random diffusion could be alleviated is through localized accumulation of high effective concentrations of signalling proteins in discrete pools in the cell (1). The inclusion of interacting proteins into liquid-liquid phase-separated (LLPS), membraneless protein droplets maintains functionally relevant proteins at high concentrations in a liquid phase at the required point of action, enhancing equilibrium binding and enzyme activity (2-6). These LLPS states have been associated with a wide range of cellular functions including regulation of signalling through, for example, nephrin (7, 8), the T-cell receptor (9), mTOR (10), and Sos-Ras (11), however, whether LLPS extends to RTK-mediated signal transduction has not been investigated. Here, we show that an RTK, fibroblast growth factor receptor 2 (FGFR2), forms a signalling competent LLPS state with two downstream effectors, a tandem Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (Shp2), and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma 1 (Plc{gamma}1). We show that these proteins assemble into a ternary complex which exploits LLPS condensation to simultaneously modulate kinase, phosphatase and phospholipase activities. Therefore, LLPS formation ensures that the requirement for prolonged, high-fidelity signalling is achieved. Additional RTKs also form LLPS with their downstream effectors, suggesting that formation of biological condensates is a key organising principle of RTK-mediated signalling, with broad implications for further mechanistic studies as well as therapeutic intervention.

cell biology↗