Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling.

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)—a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin–mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades.

No abstract

RecA is a naturally aggregating Escherichia coli protein that catalyzes the strand exchange reaction utilized in DNA repair. Previous studies have shown that the presence of salts influence RecA activity, aggregation, and stability and that salts stabilize RecA in an inverse-anionic Hofmeister series. Here we utilized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and circular dichroism (CD) to investigate how various Hofmeister salts alter the water structure and RecA solvation and aggregation. Spectroscopic studies performed in water and deuterium oxide suggest that salts alter water O-(1)H and O-(2)H stretch and bend vibrations as well as protein amide I (or I') and amide II (or II') vibrations. Anions have a much larger influence on water vibrations than cations. Water studies also show increased water-water and/or water-ion interactions in the presence of strongly hydrated SO4(2-) salts and evidence for decreased interactions with weakly hydrated Cl(-) and ClO4(-) salts. Salt-water difference infrared spectra show that kosmotropic salts are more hydrated than chaotropic salts. Interestingly, this is the opposite trend to the changes in protein solvation. Infrared spectra of RecA show that vibrations associated with protein desolvation were observed in the presence of SO4(2-) salts. Conversely, vibrations associated with protein solvation were observed in the presence of Cl(-) and ClO4(-) salts. Difference infrared studies on the dehydration of model proteins aided in identifying changes in RecA-solvent interactions. This study provides evidence that salt-induced changes in water vibrations correlate to changes in protein solvent interactions and thermal stability.

This work investigates the interactions of a series of 11 anions with caffeine by utilizing C andH NMR and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The aim of this study is to elucidate the molecular mechanisms of ion interactions with caffeine and to study how these interactions affect caffeine aggregation in aqueous solution. The chemical shift changes of caffeine C andH in the presence of salts provide a measure for anions' salting-out/salting-in abilities on individual carbon and hydrogen atoms in caffeine. The relative influences of anions on the chemical shift of individual atoms in the caffeine molecule are quantified. It is observed that strongly hydrated anions are excluded from the carbons on the six-member ring in caffeine and promote caffeine aggregation. On the other hand, weakly hydrated anions decrease caffeine aggregation by accumulating around the periphery of the caffeine molecule and binding to the ring structure. The ATR-FTIR results demonstrate that strongly hydrated anions desolvate the caffeine molecule and increase aggregation, while weakly hydrated anions have the opposite effects and salt caffeine into solution.