Showing papers by "David E. Shaw published in 2021"

Anton 3: twenty microseconds of molecular dynamics simulation before lunch

Abstract: Anton 3 is the newest member in a family of supercomputers specially designed for atomic-level simulation of molecules relevant to biology (e.g., DNA, proteins, and drug molecules). Anton 3 achieves order-of-magnitude improvements in time-to-solution over its predecessor, Anton 2 (the current state of the art), and is over 100-fold faster than any other currently available supercomputer, thereby enabling broad new avenues of research on critical questions in biology and drug discovery. This speedup means that a 512-node Anton 3 simulates a million atoms at over 100 microseconds per day. Furthermore, Anton 3 attains this performance while consuming an order of magnitude less energy per simulated microsecond than any other machine. Like its predecessors, Anton 3 was designed from the ground up around a new custom chip to best exploit the capabilities offered by new technologies. We present here the main architectural and algorithmic developments that were necessary to achieve such significant advances.

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

Abstract: 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.

A structural model of a Ras-Raf signalosome.

Abstract: 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.

Molecular basis of small-molecule binding to α-synuclein

Abstract: 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-timescale, 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.

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

Abstract: 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 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 crosslinking, 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 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. Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

Quantum chemical benchmark databases of gold-standard dimer interaction energies

Abstract: Advances in computational chemistry create an ongoing need for larger and higher-quality datasets that characterize noncovalent molecular interactions. We present three benchmark collections of quantum mechanical data, covering approximately 3,700 distinct types of interacting molecule pairs. The first collection, which we refer to as DES370K, contains interaction energies for more than 370,000 dimer geometries. These were computed using the coupled-cluster method with single, double, and perturbative triple excitations [CCSD(T)], which is widely regarded as the gold-standard method in electronic structure theory. Our second benchmark collection, a core representative subset of DES370K called DES15K, is intended for more computationally demanding applications of the data. Finally, DES5M, our third collection, comprises interaction energies for nearly 5,000,000 dimer geometries; these were calculated using SNS-MP2, a machine learning approach that provides results with accuracy comparable to that of our coupled-cluster training data. These datasets may prove useful in the development of density functionals, empirically corrected wavefunction-based approaches, semi-empirical methods, force fields, and models trained using machine learning methods. Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.13521638

Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography-High-Resolution Mass Spectrometry Analysis.

Abstract: CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Abstract: Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF), but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed, but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular Dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAF(V600E) therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical wellbeing in a stage IV colorectal cancer patient. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAF(V600E) tumors.

How does a small molecule bind at a cryptic binding site

Abstract: 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.

Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization.

Abstract: 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.

Suppressing Kv1.3 Ion Channel Activity with a Novel Small Molecule Inhibitor Ameliorates Inflammation in a Humanised Mouse Model of Ulcerative Colitis

Abstract: Background and aims The potassium channel Kv1.3 is a potentially attractive therapeutic target in T cell-mediated inflammatory diseases, as the activity of antigen-activated T cells is selectively impeded by Kv1.3 inhibition. In this study, we examined Kv1.3 as a potential therapeutic intervention point for ulcerative colitis (UC), and studied the efficacy of DES1, a small-molecule inhibitor of Kv1.3, in vitro and in vivo. Methods Kv1.3 expression on T cells in peripheral blood mononuclear cells (PBMCs) isolated from donors with and without UC was examined by flow cytometry. In biopsies from UC patients, Kv1.3-expressing CD4+ T cells were detected by flow cytometry and immunohistochemistry. In vitro, we determined the ability of DES1 to inhibit anti-CD3-driven activation of T cells. In vivo, the efficacy of DES1 was determined in a humanized mouse model of UC and compared to infliximab and tofacitinib in head-to-head studies. Results Kv1.3 expression was elevated in PBMCs from UC patients and correlated with the prevalence of TH1 and TH2 T cells. Kv1.3 expression was also detected on T cells from biopsies of UC patients. In vitro, DES1 suppressed anti-CD3-driven activation of T cells in a concentration-dependent manner. In vivo, DES1 significantly ameliorated inflammation in the UC model and most effectively so when PBMCs from donors with higher levels of activated T cells were selected for reconstitution. The efficacy of DES1 was comparable to that of either infliximab or tofacitinib. Conclusion Inhibition of Kv1.3 (by DES1, for instance) appears to be a potential therapeutic intervention strategy for UC patients.

Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex

Abstract: The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

The ΛNTON 3 ASIC: a Fire-Breathing Monster for Molecular Dynamics Simulations

Abstract: • Understand biomolecular systems through their motions •Numerical integration of Newton's laws of motion — Model atoms as point masses — Compute forces on every atom based on current positions — Update atom velocities and positions in discrete time steps of a few femtoseconds • Force computation described by a model: the force field