Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. ...Recent advances in X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and other techniques are helping us realize the dream of seeing--in atomic detail--how different parts of biomolecules shift between functional substates using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution.
Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are ...infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 β-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans β-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.
Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the ...functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory “hot spots” that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.
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► We compiled a resource of nearly 200,000 PTMs across 11 eukaryotic species ► Computational methods were developed to assign function to PTMs ► Conservation studies identify regulatory regions within domain families ► Evolutionary analysis suggests that only a fraction of PTMs are functionally important
Analysis of 200,000 PTMs across 11 species in the light of structural and interaction data reveals ground rules for establishing whether a PTM is likely to impact protein function and suggests that only a fraction of modification sites are likely to be functionally important.
Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the ...functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.
Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that ...assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.
Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site ...mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.
Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are largely unknown. ...Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrate that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.
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•Gut microbiota produce tryptamine via tryptophan (Trp) decarboxylation•Two bacterial Trp decarboxylases from gut Firmicutes were identified and characterized•Structure of one of the tryptophan decarboxylase suggests catalytic mechanism•>10% of healthy humans harbor a Trp decarboxylase in their gut microbiome
Williams et al. describe tryptophan decarboxylases from human microbiota that produce the neurotransmitter tryptamine. Although rare among bacteria more broadly, at least 10% of the human population harbors this activity in their gut community. These results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds.
Many proteins have small-molecule binding pockets that are not easily detectable in the ligand-free structures. These cryptic sites require a conformational change to become apparent; a cryptic site ...can therefore be defined as a site that forms a pocket in a holo structure, but not in the apo structure. Because many proteins appear to lack druggable pockets, understanding and accurately identifying cryptic sites could expand the set of drug targets. Previously, cryptic sites were identified experimentally by fragment-based ligand discovery and computationally by long molecular dynamics simulations and fragment docking. Here, we begin by constructing a set of structurally defined apo–holo pairs with cryptic sites. Next, we comprehensively characterize the cryptic sites in terms of their sequence, structure, and dynamics attributes. We find that cryptic sites tend to be as conserved in evolution as traditional binding pockets but are less hydrophobic and more flexible. Relying on this characterization, we use machine learning to predict cryptic sites with relatively high accuracy (for our benchmark, the true positive and false positive rates are 73% and 29%, respectively). We then predict cryptic sites in the entire structurally characterized human proteome (11,201 structures, covering 23% of all residues in the proteome). CryptoSite increases the size of the potentially “druggable” human proteome from ~40% to ~78% of disease-associated proteins. Finally, to demonstrate the utility of our approach in practice, we experimentally validate a cryptic site in protein tyrosine phosphatase 1B using a covalent ligand and NMR spectroscopy. The CryptoSite Web server is available at http://salilab.org/cryptosite.
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•Bona fide cryptic sites identified by comparison of apo and holo protein structures.•Features distinguishing cryptic sites and binding pockets identified.•Efficient and accurate prediction of cryptic sites developed.•Cryptic sites predicted for all human proteins of known structure.•The “druggable” human proteome may be larger than previously estimated.
Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5Å), providing biological phenomena with molecular descriptions. However, ...at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids - typical in a macromolecular assembly - is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.
A fundamental strategy of eukaryotic antiviral immunity involves the cGAS enzyme, which synthesizes 2′,3′-cGAMP and activates the effector STING. Diverse bacteria contain cGAS-like enzymes that ...produce cyclic oligonucleotides and induce anti-phage activity, known as CBASS. However, this activity has only been demonstrated through heterologous expression. Whether bacteria harboring CBASS antagonize and co-evolve with phages is unknown. Here, we identified an endogenous cGAS-like enzyme in Pseudomonas aeruginosa that generates 3′,3′-cGAMP during phage infection, signals to a phospholipase effector, and limits phage replication. In response, phages express an anti-CBASS protein (“Acb2”) that forms a hexamer with three 3′,3′-cGAMP molecules and reduces phospholipase activity. Acb2 also binds to molecules produced by other bacterial cGAS-like enzymes (3',3'-cUU/UA/UG/AA) and mammalian cGAS (2′,3′-cGAMP), suggesting broad inhibition of cGAS-based immunity. Upon Acb2 deletion, CBASS blocks lytic phage replication and lysogenic induction, but rare phages evade CBASS through major capsid gene mutations. Altogether, we demonstrate endogenous CBASS anti-phage function and strategies of CBASS inhibition and evasion.
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•Endogenous P. aeruginosa CBASS makes 3′,3′-cGAMP in response to phage infection•Phages inhibit CBASS via anti-CBASS (Acb2) proteins that sequester 3′,3′-cGAMP•Acb2 binds to various cyclic dinucleotides: 2′,3′-cGAMP and 3′,3′-cUU/UA/UG/AA•Diverse phages evade CBASS via mutations in their major capsid gene
Bacteriophages antagonize cGAS-like bacterial immunity by sequestering immune signaling molecules and acquiring capsid gene mutations.
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