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 EGFR's 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.

The dynamic process of ß(2)-adrenergic receptor activation.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.

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.

Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

How does a small molecule bind at a cryptic binding site?

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.

A Conserved Local Structural Motif Controls the Kinetics of PTP1B Catalysis.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin signaling pathways, making it a highly attractive target for the treatment of type II diabetes. For PTP1B to perform its enzymatic function, a loop referred to as the "WPD loop" must transition between open (catalytically incompetent) and closed (catalytically competent) conformations, which have both been resolved by X-ray crystallography. Although prior studies have established this transition as the rate-limiting step for catalysis, the transition mechanism for PTP1B and other PTPs has been unclear. Here we present an atomically detailed model of WPD loop transitions in PTP1B based on unbiased, long-timescale molecular dynamics simulations and weighted ensemble simulations. We found that a specific WPD loop region─the PDFG motif─acted as the key conformational switch, with structural changes to the motif being necessary and sufficient for transitions between long-lived open and closed states of the loop. Simulations starting from the closed state repeatedly visited open states of the loop that quickly closed again unless the infrequent conformational switching of the motif stabilized the open state. The functional importance of the PDFG motif is supported by the fact that it is well conserved across PTPs. Bioinformatic analysis shows that the PDFG motif is also conserved, and adopts two distinct conformations, in deiminases, and the related DFG motif is known to function as a conformational switch in many kinases, suggesting that PDFG-like motifs may control transitions between structurally distinct, long-lived conformational states in multiple protein families.

Discovery and Validation of the Binding Poses of Allosteric Fragment Hits to Protein Tyrosine Phosphatase 1b: From Molecular Dynamics Simulations to X-ray Crystallography.

Fragment-based drug discovery has led to six approved drugs, but the small sizes of the chemical fragments used in such methods typically result in only weak interactions between the fragment and its target molecule, which makes it challenging to experimentally determine the three-dimensional poses fragments assume in the bound state. One computational approach that could help address this difficulty is long-timescale molecular dynamics (MD) simulations, which have been used in retrospective studies to recover experimentally known binding poses of fragments. Here, we present the results of long-timescale MD simulations that we used to prospectively discover binding poses for two series of fragments in allosteric pockets on a difficult and important pharmaceutical target, protein tyrosine phosphatase 1b (PTP1b). Our simulations reversibly sampled the fragment association and dissociation process. One of the binding pockets found in the simulations has not to our knowledge been previously observed with a bound fragment, and the other pocket adopted a very rare conformation. We subsequently obtained high-resolution crystal structures of members of each fragment series bound to PTP1b, and the experimentally observed poses confirmed the simulation results. To the best of our knowledge, our findings provide the first demonstration that MD simulations can be used prospectively to determine fragment binding poses to previously unidentified pockets.

Molecular Basis of Small-Molecule Binding to α-Synuclein.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

IRAK4 dimerization and trans-autophosphorylation are induced by Myddosome assembly.

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.

Structural mechanism for Bruton's tyrosine kinase activation at the cell membrane.

Bruton's tyrosine kinase (Btk) is critical for B cell proliferation and activation, and the development of Btk inhibitors is a vigorously pursued strategy for the treatment of various B cell malignancies. A detailed mechanistic understanding of Btk activation has, however, been lacking. Here, inspired by a previous suggestion that Btk activation might depend on dimerization of its lipid-binding PH-TH module on the cell membrane, we performed long-timescale molecular dynamics simulations of membrane-bound PH-TH modules and observed that they dimerized into a single predominant conformation. We found that the phospholipid PIP3 stabilized the dimer allosterically by binding at multiple sites, and that the effects of PH-TH mutations on dimer stability were consistent with their known effects on Btk activity. Taken together, our simulation results strongly suggest that PIP3-mediated dimerization of Btk at the cell membrane is a critical step in Btk activation.

Atomic-level characterization of protein-protein association.

Despite the biological importance of protein-protein complexes, determining their structures and association mechanisms remains an outstanding challenge. Here, we report the results of atomic-level simulations in which we observed five protein-protein pairs repeatedly associate to, and dissociate from, their experimentally determined native complexes using a molecular dynamics (MD)-based sampling approach that does not make use of any prior structural information about the complexes. To study association mechanisms, we performed additional, conventional MD simulations, in which we observed numerous spontaneous association events. A shared feature of native association for these five structurally and functionally diverse protein systems was that if the proteins made contact far from the native interface, the native state was reached by dissociation and eventual reassociation near the native interface, rather than by extensive interfacial exploration while the proteins remained in contact. At the transition state (the conformational ensemble from which association to the native complex and dissociation are equally likely), the protein-protein interfaces were still highly hydrated, and no more than 20% of native contacts had formed.

Publisher Correction: Structural insights into binding specificity, efficacy and bias of a ß2AR partial agonist.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

Developing a molecular dynamics force field for both folded and disordered protein states.

Molecular dynamics (MD) simulation is a valuable tool for characterizing the structural dynamics of folded proteins and should be similarly applicable to disordered proteins and proteins with both folded and disordered regions. It has been unclear, however, whether any physical model (force field) used in MD simulations accurately describes both folded and disordered proteins. Here, we select a benchmark set of 21 systems, including folded and disordered proteins, simulate these systems with six state-of-the-art force fields, and compare the results to over 9,000 available experimental data points. We find that none of the tested force fields simultaneously provided accurate descriptions of folded proteins, of the dimensions of disordered proteins, and of the secondary structure propensities of disordered proteins. Guided by simulation results on a subset of our benchmark, however, we modified parameters of one force field, achieving excellent agreement with experiment for disordered proteins, while maintaining state-of-the-art accuracy for folded proteins. The resulting force field, a99SB-disp, should thus greatly expand the range of biological systems amenable to MD simulation. A similar approach could be taken to improve other force fields.

RNA force field with accuracy comparable to state-of-the-art protein force fields.

Molecular dynamics (MD) simulation has become a powerful tool for characterizing at an atomic level of detail the conformational changes undergone by proteins. The application of such simulations to RNA structures, however, has proven more challenging, due in large part to the fact that the physical models ("force fields") available for MD simulations of RNA molecules are substantially less accurate in many respects than those currently available for proteins. Here, we introduce an extensive revision of a widely used RNA force field in which the parameters have been modified, based on quantum mechanical calculations and existing experimental information, to more accurately reflect the fundamental forces that stabilize RNA structures. We evaluate these revised parameters through long-timescale MD simulations of a set of RNA molecules that covers a wide range of structural complexity, including single-stranded RNAs, RNA duplexes, RNA hairpins, and riboswitches. The structural and thermodynamic properties measured in these simulations exhibited dramatically improved agreement with experimentally determined values. Based on the comparisons we performed, this RNA force field appears to achieve a level of accuracy comparable to that of state-of-the-art protein force fields, thus significantly advancing the utility of MD simulation as a tool for elucidating the structural dynamics and function of RNA molecules and RNA-containing biological assemblies.

Mechanism of Coupled Folding-upon-Binding of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs), which in isolation do not adopt a well-defined tertiary structure but instead populate a structurally heterogeneous ensemble of interconverting states, play important roles in many biological pathways. IDPs often fold into ordered states upon binding to their physiological interaction partners (a so-called "folding-upon-binding" process), but it has proven difficult to obtain an atomic-level description of the structural mechanisms by which they do so. Here, we describe in atomic detail the folding-upon-binding mechanism of an IDP segment to its binding partner, as observed in unbiased molecular dynamics simulations. In our simulations, we observed over 70 binding and unbinding events between the α-helical molecular recognition element (α-MoRE) of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the measles virus phosphoprotein complex. We found that folding-upon-binding primarily occurred through induced-folding pathways (in which intermolecular contacts form before or concurrently with the secondary structure of the disordered protein)-an observation supported by previous experiments-and that the transition state ensemble was characterized by formation of just a few key intermolecular contacts and was otherwise highly structurally heterogeneous. We found that when a large amount of helical content was present early in a transition path, NTAIL typically unfolded and then refolded after additional intermolecular contacts formed. We also found that, among conformations with similar numbers of intermolecular contacts, those with less helical content had a higher probability of ultimately forming the native complex than conformations with more helical content, which were more likely to unbind. These observations suggest that even after intermolecular contacts have formed, disordered regions can have a kinetic advantage over folded regions in the folding-upon-binding process.

Structural insights into binding specificity, efficacy and bias of a ß2AR partial agonist.

Salmeterol is a partial agonist for the ß2 adrenergic receptor (ß2AR) and the first long-acting ß2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound ß2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between ß1AR and ß2AR explain the high receptor-subtype selectivity. A structural comparison with the ß2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited ß-arrestin recruitment for salmeterol.

AutoPH4: An Automated Method for Generating Pharmacophore Models from Protein Binding Pockets.

Pharmacophore models are widely used in computational drug discovery (e.g., in the virtual screening of drug molecules) to capture essential information about interactions between ligands and a target protein. Generating pharmacophore models from protein structures is typically a manual process, but there has been growing interest in automated pharmacophore generation methods. Automation makes feasible the processing of large numbers of protein conformations, such as those generated by molecular dynamics (MD) simulations, and thus may help achieve the longstanding goal of incorporating protein flexibility into virtual screening workflows. Here, we present AutoPH4, a new automated method for generating pharmacophore models based on protein structures; we show that a virtual screening workflow incorporating AutoPH4 ranks compounds more accurately than any other pharmacophore-based virtual screening workflow for which results on a public benchmark have been reported. The strong performance of the virtual screening workflow indicates that the AutoPH4 component of the workflow generates high-quality pharmacophores, making AutoPH4 promising for use in future virtual screening workflows as well, such as ones that use conformations generated by MD simulations.