Anton Arkhipov

Architecture and Membrane Interactions of the EGF Receptor

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to 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 EGFRs intracellular module with the membrane are critical in maintaining this coupling.

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

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. Although 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.

Oncogenic Mutations Counteract Intrinsic Disorder in the EGFR Kinase and Promote Receptor Dimerization

The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.

Transitions to catalytically inactive conformations in EGFR kinase

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called “Src-like inactive” conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed “extended” conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another [“Asp-Phe-Gly-out” (“DFG-out”)] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or “cracking,” which supports a prediction of energy landscape theory. We used hydrogen–deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.

Membrane Interaction of Bound Ligands Contributes to the Negative Binding Cooperativity of the EGF Receptor

The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo.

Inferring cortical function in the mouse visual system through large-scale systems neuroscience

The scientific mission of the Project MindScope is to understand neocortex, the part of the mammalian brain that gives rise to perception, memory, intelligence, and consciousness. We seek to quantitatively evaluate the hypothesis that neocortex is a relatively homogeneous tissue, with smaller functional modules that perform a common computational function replicated across regions. We here focus on the mouse as a mammalian model organism with genetics, physiology, and behavior that can be readily studied and manipulated in the laboratory. We seek to describe the operation of cortical circuitry at the computational level by comprehensively cataloging and characterizing its cellular building blocks along with their dynamics and their cell type-specific connectivities. The project is also building large-scale experimental platforms (i.e., brain observatories) to record the activity of large populations of cortical neurons in behaving mice subject to visual stimuli. A primary goal is to understand the series of operations from visual input in the retina to behavior by observing and modeling the physical transformations of signals in the corticothalamic system. We here focus on the contribution that computer modeling and theory make to this long-term effort.

Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family

The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human. DOI: http://dx.doi.org/10.7554/eLife.00708.001

Classification of electrophysiological and morphological types in mouse visual cortex

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To profile morpho-electric properties of mammalian neurons systematically, we established a single cell characterization pipeline using standardized patch clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly-accessible online database, the Allen Cell Types Database, to display these data sets. Intrinsic physiological and morphological properties were measured from over 1,800 neurons from the adult laboratory mouse visual cortex. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We establish a taxonomy of morphologically- and electrophysiologically-defined cell types for this region of cortex with 17 e-types and 35 m-types, as well as an initial correspondence with previously-defined transcriptomic cell types using the same transgenic mouse lines.

Visual physiology of the Layer 4 cortical circuit in silico

Despite advances in experimental techniques and accumulation of large datasets concerning the composition and properties of the cortex, quantitative modeling of cortical circuits under in-vivo-like conditions remains challenging. Here we report and publicly release a biophysically detailed circuit model of layer 4 in the mouse primary visual cortex, receiving thalamo-cortical visual inputs. The 45,000-neuron model was subjected to a battery of visual stimuli, and results were compared to published work and new in vivo experiments. Simulations reproduced a variety of observations, including effects of optogenetic perturbations. Critical to the agreement between responses in silico and in vivo were the rules of functional synaptic connectivity between neurons. Interestingly, after extreme simplification the model still performed satisfactorily on many measurements, although quantitative agreement with experiments suffered. These results emphasize the importance of functional rules of cortical wiring and enable a next generation of data-driven models of in vivo neural activity and computations. AUTHOR SUMMARY How can we capture the incredible complexity of brain circuits in quantitative models, and what can such models teach us about mechanisms underlying brain activity? To answer these questions, we set out to build extensive, bio-realistic models of brain circuitry employing systematic datasets on brain structure and function. Here we report the first modeling results of this project, focusing on the layer 4 of the primary visual cortex (V1) of the mouse. Our simulations reproduced a variety of experimental observations in a large battery of visual stimuli. The results elucidated circuit mechanisms determining patters of neuronal activity in layer 4 – in particular, the roles of feedforward thalamic inputs and specific patterns of intracortical connectivity in producing tuning of neuronal responses to the orientation of motion. Simplification of neuronal models led to specific deficiencies in reproducing experimental data, giving insights into how biological details contribute to various aspects of brain activity. To enable future development of more sophisticated models, we make the software code, the model, and simulation results publicly available.