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Supplementary Methods and Materials from Equally Potent Inhibition of c-Src and Abl by Compounds that Recognize Inactive Kinase Conformations

Supplementary Table 1 from Equally Potent Inhibition of c-Src and Abl by Compounds that Recognize Inactive Kinase Conformations

Supplementary Figures 1-2 from Equally Potent Inhibition of c-Src and Abl by Compounds that Recognize Inactive Kinase Conformations

Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia. Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2,000-fold lower. Imatinib recognition requires a specific inactive conformation of the kinase domain, in which a conserved Asp-Phe-Gly (DFG) motif is flipped with respect to the active conformation. The inability of c-Src to readily adopt this flipped DFG conformation was thought to underlie the selectivity of imatinib for Abl over c-Src. Here, we present a series of inhibitors (DSA compounds) that are based on the core scaffold of imatinib but which bind with equally high potency to c-Src and Abl. The DSA compounds bind to c-Src in the DFG-flipped conformation, as confirmed by crystal structures and kinetic analysis. The origin of the high affinity of these compounds for c-Src is suggested by the fact that they also inhibit clinically relevant Abl variants bearing mutations in a structural element, the P-loop, that normally interacts with the phosphate groups of ATP but is folded over a substructure of imatinib in Abl. Importantly, several of the DSA compounds block the growth of Ba/F3 cells harboring imatinib-resistant BCR-ABL mutants, including the Thr315Ile “gatekeeper” mutation, but do not suppress the growth of parental Ba/F3 cells. [Cancer Res 2009;69(6):2384–92

Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a v-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems.

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs., Bone morphogenetic protein (BMP) receptors are single pass transmembrane serine/threonine kinases that form tetrameric complexes comprised of two type I and two type II BMP receptors. Here the authors characterize a structure of an active type I/type II kinase tetramer providing insight into molecular mechanism driving ligand-induced signaling.

Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations, we showed that RND1 reinforces the plexin dimerization interface, whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase-binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

Plexins are semaphorin receptors that play essential roles in neuronal axon guidance and in many other important biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism, and is modulated by small signaling GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations we showed that RND1 reinforces plexin dimerization interface whereas RhoD destabilizes it due to their differential interaction with cell membrane. Upon binding plexin dimers at the Rho-GTPase binding (RBD) domains, RND1 and RhoD interact differently with the inner leaflet of cell membrane, and exert opposite effects on the dimerization interface via an allosteric network involving the RBD domain, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively-charged membrane interface.

The protein JAK2 is a prototypical member of the Janus kinase family, and mediates signals from numerous cytokine receptors. The constitutively active V617F mutant of JAK2 is prevalent in many bone marrow disorders, blood cancers, and autoimmune diseases, and is an important drug target. Structures have been determined for each of the four individual domains making up JAK2, and for certain pairs of these domains, but no structure of full-length JAK2 is available, and thus the mechanisms underlying JAK2 regulation and the aberrant activity of the V617F mutant have been incompletely understood. Here we propose structural models of full-length JAK2 in both its active and inactive forms. Construction of these models was informed by long-timescale molecular dynamics simulations. Subsequent mutagenesis experiments showed that mutations at the putative interdomain interfaces modulated JAK2 activity. The models provide a structural basis for understanding JAK2 autoinhibition and activation, and suggest that the constitutive activity of the V617F mutant may arise from a dual effect of destabilizing the inactive conformation and stabilizing the active conformation.

Trans-autophosphorylation is among the most prevalent means of protein kinase activation, yet its molecular basis is poorly defined. In Toll-like receptor and interleukin-1 receptor signaling pathways, the kinase IRAK4 is recruited to the membrane-proximal adaptor MyD88 through death domain (DD) interactions, forming the oligomeric Myddosome and mediating NF-κB activation. Here we show that unphosphorylated IRAK4 dimerizes in solution with a KD of 2.5 μM and that Myddosome assembly greatly enhances IRAK4 kinase domain (KD) autophosphorylation at sub-KD concentrations. The crystal structure of the unphosphorylated IRAK4(KD) dimer captures a conformation that appears to represent the actual trans-autophosphorylation reaction, with the activation loop phosphosite of one IRAK4 monomer precisely positioned for phosphotransfer by its partner. We show that dimerization is crucial for IRAK4 autophosphorylation in vitro and ligand-dependent signaling in cells. These studies identify a mechanism for oligomerization-driven allosteric autoactivation of IRAK4 that may be general to other kinases activated by autophosphorylation.

SummaryHow 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.

The protein tyrosine kinase Jak2 mediates signaling through numerous cytokine receptors. Jak2 possesses a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). Through unknown mechanisms, JH2 regulates the catalytic activity of JH1, and hyperactivating mutations in the JH2 region of human Jak2 are causative for myeloproliferative neoplasms (MPNs). We showed previously that Jak2 JH2 is in fact catalytically active. Here, we present crystal structures of human Jak2 JH2, both wild-type and the most prevalent MPN mutant, V617F. The structures reveal that JH2 adopts the fold of a prototypical protein kinase but binds Mg-ATP non-canonically. The structural and biochemical data indicate that the V617F mutation rigidifies α-helix C in the N lobe of JH2, which facilitates trans-phosphorylation of JH1. The crystal structures of JH2 afford new opportunities for the design of novel Jak2 therapeutics targeting MPNs.

Following Folding Fast Many protein functions involve conformational changes that occur on time-scales between tens of microseconds and milliseconds. This has limited the usefulness of all-atom molecular dynamics simulations, which are performed over shorter time-scales. Shaw et al. (p. 341 ) now report millisecond-scale, all-atom molecular dynamics simulations in an explicitly represented solvent environment. Simulation of the folding of a WW domain showed a well-defined folding pathway and simulation of the dynamics of bovine pancreatic trypsin inhibitor showed interconversion between distinct conformational states.

Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia. Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2,000-fold lower. Imatinib recognition requires a specific inactive conformation of the kinase domain, in which a conserved Asp-Phe-Gly (DFG) motif is flipped with respect to the active conformation. The inability of c-Src to readily adopt this flipped DFG conformation was thought to underlie the selectivity of imatinib for Abl over c-Src. Here, we present a series of inhibitors (DSA compounds) that are based on the core scaffold of imatinib but which bind with equally high potency to c-Src and Abl. The DSA compounds bind to c-Src in the DFG-flipped conformation, as confirmed by crystal structures and kinetic analysis. The origin of the high affinity of these compounds for c-Src is suggested by the fact that they also inhibit clinically relevant Abl variants bearing mutations in a structural element, the P-loop, that normally interacts with the phosphate groups of ATP but is folded over a substructure of imatinib in Abl. Importantly, several of the DSA compounds block the growth of Ba/F3 cells harboring imatinib-resistant BCR-ABL mutants, including the Thr315Ile “gatekeeper” mutation, but do not suppress the growth of parental Ba/F3 cells. [Cancer Res 2009;69(6):2384–92

Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity., Protein tyrosine kinases are subject to multiple regulatory mechanisms. Foda et al. show that reactants and products of the tyrosine kinase Src bind its catalytic domain with opposite cooperativity, and identify an allosteric network of dynamically coupled amino acids that underlie this behaviour.

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site’s location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001, eLife digest Hormones are chemicals that are produced to carry signals around the body. Mammals, including humans, need hormones called insulin and insulin-like growth factors (or IGF for short) to grow and develop normally. These hormones bind to, and activate, specific proteins—known as receptors—that span from the outside of the cell to the inside through the cell's surface membrane. The insulin and IGF receptors are complex molecules, each composed of two identical protein subunits that are linked together. The hormones bind to the part of the proteins (known as the extracellular domains) that are on the outside surface of cells. When no hormone is bound to the receptor, these two extracellular domains form an inverted ‘V’ shape and the two regions that cross the membrane are held far apart. It was unclear, however, how this shape changes when the hormone binds, and how this change in shape activates the receptor. Kavran et al. have now compared the known three-dimensional structures of the extracellular domains of the insulin receptor, both with and without a molecule of insulin bound to it. This comparison highlighted an interaction between the two receptor subunits that was disrupted when insulin was bound to the receptor. This interaction appeared to stabilize the inverted ‘V’ structure and hold apart the parts of the proteins that span the surface—which in turn separates the regions of the proteins that are inside the cell. Kavran et al. suggest that this separation keeps the insulin receptor in an inactive state. Removing either the whole extracellular domain of the IGF receptor—or a smaller portion that keeps the regions that cross the cell membrane separate—resulted in the receptor being activated, even when insulin-like growth factor was not bound to the receptor. Kavran et al. then confirmed that when a molecule of insulin-like growth factor binds to the extracellular domain of the IGF receptor, it causes a shape change that moves the parts of the receptor that span the cell membrane closer together. This results in the regions of the receptor proteins located inside the cell adding chemical tags, called phosphate groups, to one another—which activates the receptor. When there are problems with how these receptors are activated or inactivated in humans, serious disorders including diabetes and cancer can occur. As such, the findings of Kavran et al. might help future work aimed at regulating the activation of the insulin and IGF receptors to treat these and other diseases. DOI: http://dx.doi.org/10.7554/eLife.03772.002

Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using unbiased molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.

The human epidermal growth factor receptor (HER) tyrosine kinases homo- and heterodimerize to activate downstream signaling pathways. HER3 is a catalytically impaired member of the HER family that contributes to the development of several human malignancies and is mutated in a subset of cancers. HER3 signaling depends on heterodimerization with a catalytically active partner, in particular epidermal growth factor receptor (EGFR) (the founding family member, also known as HER1) or HER2. The activity of homodimeric complexes of catalytically active HER family members depends on allosteric activation between the two kinase domains. To determine the structural basis for HER3 signaling through heterodimerization with a catalytically active HER family member, we solved the crystal structure of the heterodimeric complex formed by the isolated kinase domains of EGFR and HER3. The structure showed HER3 as an allosteric activator of EGFR and revealed a conserved role of the allosteric mechanism in activation of HER family members through heterodimerization. To understand the effects of cancer-associated HER3 mutations at the molecular level, we solved the structures of two kinase domains of HER3 mutants, each in a heterodimeric complex with the kinase domain of EGFR. These structures, combined with biochemical analysis and molecular dynamics simulations, indicated that the cancer-associated HER3 mutations enhanced the allosteric activator function of HER3 by redesigning local interactions at the dimerization interface.

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., Author Summary Epidermal growth factor receptor (EGFR) molecules are of central importance in cellular communication. Embedded in the cell membrane, these receptors bind epidermal growth factor (EGF) molecules outside the cell and translate this binding into specific biochemical signals inside the cell, which in turn trigger cell proliferation, migration, or differentiation. EGFR dysfunction has been implicated in a variety of cancers, and EGFR-targeting drugs are commonly used in cancer treatments. It has been widely assumed that the extracellular portion of an EGFR molecule protrudes perpendicularly from the cell membrane. In detailed, atomic-level computer simulations, however, we find that it lies down on the membrane, placing its EGF-binding site adjacent to the membrane surface. We further show that EGF may interact with EGFR in two distinct ways (with or without the involvement of the membrane). This may explain the experimental finding that an EGF molecule binds to EGFR more weakly at higher EGF concentration. This phenomenon, which is a manifestation of an underlying negative cooperativity, is an important but poorly understood characteristic of EGFR activity. In this study, we also model and analyze the glycan chains attached to EGFR, which are integral to its behavior in living cells.

How drugs bind to their receptors—from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or “pose”—has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the β 1 - and β 2 -adrenergic receptors, initially making contact with a vestibule on each receptor’s extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug–receptor binding and unbinding rates.

SummaryThe 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.

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N-lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family.

In many protein kinases, a characteristic conformational change (the “DFG flip”) connects catalytically active and inactive conformations. Many kinase inhibitors—including the cancer drug imatinib—selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomic-level detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.

Na + /H + antiporters are central to cellular salt and pH homeostasis. The structure of Escherichia coli NhaA was recently determined, but its mechanisms of transport and pH regulation remain elusive. We performed molecular dynamics simulations of NhaA that, with existing experimental data, enabled us to propose an atomically detailed model of antiporter function. Three conserved aspartates are key to our proposed mechanism: Asp 164 (D164) is the Na + -binding site, D163 controls the alternating accessibility of this binding site to the cytoplasm or periplasm, and D133 is crucial for pH regulation. Consistent with experimental stoichiometry, two protons are required to transport a single Na + ion: D163 protonates to reveal the Na + -binding site to the periplasm, and subsequent protonation of D164 releases Na + . Additional mutagenesis experiments further validated the model.

The ability to perform long, accurate molecular dynamics (MD) simulations involving proteins and other biological macro-molecules could in principle provide answers to some of the most important currently outstanding questions in the fields of biology, chemistry and medicine. A wide range of biologically interesting phenomena, however, occur over time scales on the order of a millisecond--about three orders of magnitude beyond the duration of the longest current MD simulations. In this paper, we describe a massively parallel machine called Anton, which should be capable of executing millisecond-scale classical MD simulations of such biomolecular systems. The machine, which is scheduled for completion by the end of 2008, is based on 512 identical MD-specific ASICs that interact in a tightly coupled manner using a specialized high-speed communication network. Anton has been designed to use both novel parallel algorithms and special-purpose logic to dramatically accelerate those calculations that dominate the time required for a typical MD simulation. The remainder of the simulation algorithm is executed by a programmable portion of each chip that achieves a substantial degree of parallelism while preserving the flexibility necessary to accommodate anticipated advances in physical models and simulation methods.