Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs.

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

Partitioning of an Enzyme-Polymer Surfactant Nanocomplex into Lipid-Rich Cellular Compartments Drives In Situ Hydrolysis of Organophosphates.

Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.

Biophysical characterization of the ETV6 PNT domain polymerization interfaces.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01.

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2-UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

BAlaS: fast, interactive and accessible computational alanine-scanning using BudeAlaScan.

MOTIVATION: In experimental protein engineering, alanine-scanning mutagenesis involves the replacement of selected residues with alanine to determine the energetic contribution of each side chain to forming an interaction. For example, it is often used to study protein-protein interactions. However, such experiments can be time-consuming and costly, which has led to the development of programmes for performing computational alanine-scanning mutagenesis (CASM) to guide experiments. While programmes are available for this, there is a need for a real-time web application that is accessible to non-expert users. RESULTS: Here, we present BAlaS, an interactive web application for performing CASM via BudeAlaScan and visualizing its results. BAlaS is interactive and intuitive to use. Results are displayed directly in the browser for the structure being interrogated enabling their rapid inspection. BAlaS has broad applications in areas, such as drug discovery and protein-interface design. AVAILABILITY AND IMPLEMENTATION: BAlaS works on all modern browsers and is available through the following website: https://balas.app. The project is open source, distributed using an MIT license and is available on GitHub (https://github.com/wells-wood-research/balas).

The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli.

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein*.

We investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS-CoV-2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor-binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA-approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitamin D); retinoids (some known to be active in vitro, and vitamin A); and vitamin K as potential ligands that may stabilize the closed conformation. The SARS-CoV-2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.

Stapled Peptides as HIF-1α/p300 Inhibitors: Helicity Enhancement in the Bound State Increases Inhibitory Potency.

Protein-protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide-based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia-inducible factor 1 (HIF-1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF-1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α-helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α-helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra.

Novel Anti-Inflammatory Peptides Based on Chemokine-Glycosaminoglycan Interactions Reduce Leukocyte Migration and Disease Severity in a Model of Rheumatoid Arthritis.

Inflammation is characterized by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins that interact with leukocytes to initiate their activation and extravasation via chemokine receptors. This is enabled through chemokine immobilization by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C terminus, interacts with the negatively charged GAGs, which is considered an essential interaction for the chemokine function. Short-chain peptides based on this GAG-binding region of the chemokines CCL5, CXCL8, and CXCL12γ were synthesized using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity, which translated into a reduction of leukocyte migration across a cultured human endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulfate from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis tissue. The data suggest that the peptide competes with the wild-type chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the proinflammatory cytokine TNF-α in a murine Ag-induced arthritis model. Taken together, evidence is provided for interfering with the chemokine-GAG interaction as a relevant therapeutic approach.

Beyond icosahedral symmetry in packings of proteins in spherical shells.

The formation of quasi-spherical cages from protein building blocks is a remarkable self-assembly process in many natural systems, where a small number of elementary building blocks are assembled to build a highly symmetric icosahedral cage. In turn, this has inspired synthetic biologists to design de novo protein cages. We use simple models, on multiple scales, to investigate the self-assembly of a spherical cage, focusing on the regularity of the packing of protein-like objects on the surface. Using building blocks, which are able to pack with icosahedral symmetry, we examine how stable these highly symmetric structures are to perturbations that may arise from the interplay between flexibility of the interacting blocks and entropic effects. We find that, in the presence of those perturbations, icosahedral packing is not the most stable arrangement for a wide range of parameters; rather disordered structures are found to be the most stable. Our results suggest that (i) many designed, or even natural, protein cages may not be regular in the presence of those perturbations and (ii) optimizing those flexibilities can be a possible design strategy to obtain regular synthetic cages with full control over their surface properties.

A General Mechanism for Signal Propagation in the Nicotinic Acetylcholine Receptor Family.

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic activity in the central nervous system. The α7 subtype, in particular, has attracted considerable interest in drug discovery as a target for several conditions, including Alzheimer's disease and schizophrenia. Identifying agonist-induced structural changes underlying nAChR activation is fundamentally important for understanding biological function and rational drug design. Here, extensive equilibrium and nonequilibrium molecular dynamics simulations, enabled by cloud-based high-performance computing, reveal the molecular mechanism by which structural changes induced by agonist unbinding are transmitted within the human α7 nAChR. The simulations reveal the sequence of coupled structural changes involved in driving conformational change responsible for biological function. Comparison with simulations of the α4ß2 nAChR subtype identifies features of the dynamical architecture common to both receptors, suggesting a general structural mechanism for signal propagation in this important family of receptors.

Engineering protein stability with atomic precision in a monomeric miniprotein.

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (TM) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the TM to 20 °C. We attribute this result to the loss of CH-π interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

Class II chaperonins are essential multisubunit complexes that aid the folding of nonnative proteins in the cytosol of archaea and eukarya. They use energy derived from ATP to drive a series of structural rearrangements that enable polypeptides to fold within their central cavity. These events are regulated by an elaborate allosteric mechanism in need of elucidation. We employed mutagenesis and experimental analysis in concert with in silico molecular dynamics simulations and interface-binding energy calculations to investigate the class II chaperonin from Thermoplasma acidophilum. Here we describe the effects on the asymmetric allosteric mechanism and on hetero-oligomeric complex formation in a panel of mutants in the ATP-binding pocket of the α and ß subunits. Our observations reveal a potential model for a nonconcerted folding mechanism optimized for protecting and refolding a range of nonnative substrates under different environmental conditions, starting to unravel the role of subunit heterogeneity in this folding machine and establishing important links with the behavior of the most complex eukaryotic chaperonins.-Shoemark, D. K., Sessions, R. B., Brancaccio, A., Bigotti, M. G. Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

The Aminotriazole Antagonist Cmpd-1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor.

The widely expressed G-protein coupled receptors (GPCRs) are versatile signal transducer proteins that are attractive drug targets but structurally challenging to study. GPCRs undergo a number of conformational rearrangements when transitioning from the inactive to the active state but have so far been believed to adopt a fairly conserved inactive conformation. Using 19 F NMR spectroscopy and advanced molecular dynamics simulations we describe a novel inactive state of the adenosine 2A receptor which is stabilised by the aminotriazole antagonist Cmpd-1. We demonstrate that the ligand stabilises a unique conformation of helix V and present data on the putative binding mode of the compound involving contacts to the transmembrane bundle as well as the extracellular loop 2.

Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation.

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.

ISAMBARD: an open-source computational environment for biomolecular analysis, modelling and design.

MOTIVATION: The rational design of biomolecules is becoming a reality. However, further computational tools are needed to facilitate and accelerate this, and to make it accessible to more users. RESULTS: Here we introduce ISAMBARD, a tool for structural analysis, model building and rational design of biomolecules. ISAMBARD is open-source, modular, computationally scalable and intuitive to use. These features allow non-experts to explore biomolecular design in silico. ISAMBARD addresses a standing issue in protein design, namely, how to introduce backbone variability in a controlled manner. This is achieved through the generalization of tools for parametric modelling, describing the overall shape of proteins geometrically, and without input from experimentally determined structures. This will allow backbone conformations for entire folds and assemblies not observed in nature to be generated de novo, that is, to access the 'dark matter of protein-fold space'. We anticipate that ISAMBARD will find broad applications in biomolecular design, biotechnology and synthetic biology. AVAILABILITY AND IMPLEMENTATION: A current stable build can be downloaded from the python package index (https://pypi.python.org/pypi/isambard/) with development builds available on GitHub (https://github.com/woolfson-group/) along with documentation, tutorial material and all the scripts used to generate the data described in this paper. CONTACT: d.n.woolfson@bristol.ac.uk or chris.wood@bristol.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The dynamical interplay between a megadalton peptide nanocage and solutes probed by microsecond atomistic MD; implications for design.

Understanding the assembly and dynamics of protein-based supramolecular capsids and cages is of fundamental importance and could lead to applications in synthetic biology and biotechnology. Here we present long and large atomistic molecular dynamics simulations of de novo designed self-assembling protein nanocages (SAGEs) in aqueous media. Microsecond simulations, comprised of ≈42 million atoms for three pre-formed SAGEs of different charges, in the presence of solutes and solvent have been completed. Here, the dynamics, stability and porosity of the peptide networks are explored along with their interactions with ions, small molecules and macromolecular solutes. All assemblies are stable over the µs timescale, and the solutes show a mixture of transport behaviour across or adherence to the fabric of the SAGE particles. Solute proteins largely retained native-like conformation on contact with SAGE. Certain residues of the SAGE peptides are identified as "repeat offenders" for contacting many different solutes, which suggest modifications to reduce non-specific binding. These studies highlight how molecular dynamics can aid the design process of SAGE and similar assemblies for potential applications as diverse as platforms for drug and vaccine delivery and nanoreactors to encapsulate enzyme pathways.