Summary How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. While the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Summary Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is a key step in cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role, but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N-termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N-termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR’s intracellular module with the membrane were found to be critical in maintaining this coupling.

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a new structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.

Bioorthogonal catalysis broadens the functional possibilities of intracellular chemistry. Effective delivery and regulation of synthetic catalytic systems in cells is challenging due to the complex intracellular environment and catalyst instability. Here, we report the fabrication of protein-sized bioorthogonal nanozymes through the encapsulation of hydrophobic transition metal catalysts into the monolayer of water-soluble gold nanoparticles. The activity of these catalysts can be reversibly controlled by binding a supramolecular cucurbit[7]uril ‘gate-keeper’ onto the monolayer surface, providing a biomimetic control mechanism that mimics the allosteric regulation of enzymes. The potential of this gated nanozyme for use in imaging and therapeutic applications was demonstrated through triggered cleavage of allylcarbamates for pro-fluorophore activation and propargyl groups for prodrug activation inside living cells.

Correlation of the surface physicochemical properties of nanoparticles with their interactions with biosystems provides key foundational data for nanomedicine. We report here the systematic synthesis of 2, 4, and 6 nm core gold nanoparticles (AuNP) featuring neutral (zwitterionic), anionic, and cationic headgroups. The cellular internalization of these AuNPs was quantified, providing a parametric evaluation of charge and size effects. Contrasting behavior was observed with these systems: with zwitterionic and anionic particles, uptake decreased with increasing AuNP size, whereas with cationic particles uptake increased with increasing particle size. Through mechanistic studies of the uptake process we can attribute these opposing trends to a surface-dictated shift in uptake pathways. Zwitterionic NPs are primarily internalized through passive diffusion, while the internalization of cationic and anionic NPs is dominated by multiple endocytic pathways. Our study demonstrates that size and surface charge interact in an interrelated fashion to modulate nanoparticle uptake into cells, providing an engineering tool for designing nanomaterials for specific biological applications.

EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (β2-β3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.

Human serum albumin (HSA) inhibits the formation of amyloid beta-peptide (Abeta) fibrils in human plasma. However, currently it is not known how HSA affects the formation of the highly toxic soluble diffusible oligomers that occur in the initial stages of Abeta fibrillization. We have therefore investigated by solution NMR the interaction of HSA with the Abeta(12-28) peptide, which has been previously shown to provide a reliable and stable model for the early prefibrillar oligomers as well as to contain key determinants for the recognition by albumin. For this purpose we propose a novel NMR approach based on the comparative analysis of Abeta in its inhibited and filtrated states monitored through both saturation transfer difference and recently developed nonselective off-resonance relaxation experiments. This combined NMR strategy reveals a mechanism for the oligomerization inhibitory function of HSA, according to which HSA targets preferentially the soluble oligomers of Abeta(12-28) rather than its monomeric state. Specifically, HSA caps the exposed hydrophobic patches located at the growing and/or transiently exposed sites of the Abeta oligomers, thereby blocking the addition of further monomers and the growth of the prefibrillar assemblies. The proposed model has implications not only for the pharmacological treatment of Alzheimer's disease specifically but also for the inhibition of oligomerization in amyloid-related diseases in general. In addition, the proposed NMR approach is expected to be useful for the investigation of the mechanism of action of other oligomerization inhibitors as well as of other amyloidogenic systems.

Early detection of biofilms is crucial for limiting infection-based damage. Imaging these biofilms is challenging: conventional imaging agents are unable to penetrate the dense matrix of the biofilm, and many imaging agents are susceptible to false positive/negative responses due to phenotypical mutations of the constituent microbes. We report the creation of pH-responsive nanoparticles with embedded transition metal catalysts (nanozymes) that effectively target the acidic microenvironment of biofilms. These pH-switchable nanozymes generate imaging agents through bioorthogonal activation of profluorophores inside biofilms. The specificity of these nanozymes for imaging biofilms in complex biosystems was demonstrated using coculture experiments.