ZAP-70 in Signaling, Biology, and Disease.

T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70.

The Roots of Genetic Coding in Aminoacyl-tRNA Synthetase Duality.

Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.

An Element of Life.

While it has been known for decades that the essential function of selenium was in the form of its incorporation as selenocysteine into selenoproteins-including the enzyme glutathione peroxidase-4-now, Ingold et al. (2018) reveal the precise role of selenolate-based catalysis by this enzyme.

Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

Site-Specific Self-Catalyzed DNA Depurination: A Biological Mechanism That Leads to Mutations and Creates Sequence Diversity.

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

Sortase A: A Model for Transpeptidation and Its Biological Applications.

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

Control of proton transport and hydrogenation in double-gated graphene.

The basal plane of graphene can function as a selective barrier that is permeable to protons1,2 but impermeable to all ions3,4 and gases5,6, stimulating its use in applications such as membranes1,2,7,8, catalysis9,10 and isotope separation11,12. Protons can chemically adsorb on graphene and hydrogenate it13,14, inducing a conductor-insulator transition that has been explored intensively in graphene electronic devices13-17. However, both processes face energy barriers1,12,18 and various strategies have been proposed to accelerate proton transport, for example by introducing vacancies4,7,8, incorporating catalytic metals1,19 or chemically functionalizing the lattice18,20. But these techniques can compromise other properties, such as ion selectivity21,22 or mechanical stability23. Here we show that independent control of the electric field, E, at around 1 V nm-1, and charge-carrier density, n, at around 1 × 1014 cm-2, in double-gated graphene allows the decoupling of proton transport from lattice hydrogenation and can thereby accelerate proton transport such that it approaches the limiting electrolyte current for our devices. Proton transport and hydrogenation can be driven selectively with precision and robustness, enabling proton-based logic and memory graphene devices that have on-off ratios spanning orders of magnitude. Our results show that field effects can accelerate and decouple electrochemical processes in double-gated 2D crystals and demonstrate the possibility of mapping such processes as a function of E and n, which is a new technique for the study of 2D electrode-electrolyte interfaces.

Alkene dialkylation by triple radical sorting.

The development of bimolecular homolytic substitution (SH2) catalysis has expanded cross-coupling chemistries by enabling the selective combination of any primary radical with any secondary or tertiary radical through a radical sorting mechanism1-8. Biomimetic9,10 SH2 catalysis can be used to merge common feedstock chemicals-such as alcohols, acids and halides-in various permutations for the construction of a single C(sp3)-C(sp3) bond. The ability to sort these two distinct radicals across commercially available alkenes in a three-component manner would enable the simultaneous construction of two C(sp3)-C(sp3) bonds, greatly accelerating access to complex molecules and drug-like chemical space11. However, the simultaneous in situ formation of electrophilic and primary nucleophilic radicals in the presence of unactivated alkenes is problematic, typically leading to statistical radical recombination, hydrogen atom transfer, disproportionation and other deleterious pathways12,13. Here we report the use of bimolecular homolytic substitution catalysis to sort an electrophilic radical and a nucleophilic radical across an unactivated alkene. This reaction involves the in situ formation of three distinct radical species, which are then differentiated by size and electronics, allowing for regioselective formation of the desired dialkylated products. This work accelerates access to pharmaceutically relevant C(sp3)-rich molecules and defines a distinct mechanistic approach for alkene dialkylation.

Selective lignin arylation for biomass fractionation and benign bisphenols.

Lignocellulose is mainly composed of hydrophobic lignin and hydrophilic polysaccharide polymers, contributing to an indispensable carbon resource for green biorefineries1,2. When chemically treated, lignin is compromised owing to detrimental intra- and intermolecular crosslinking that hampers downstream process3,4. The current valorization paradigms aim to avoid the formation of new C-C bonds, referred to as condensation, by blocking or stabilizing the vulnerable moieties of lignin5-7. Although there have been efforts to enhance biomass utilization through the incorporation of phenolic additives8,9, exploiting lignin's proclivity towards condensation remains unproven for valorizing both lignin and carbohydrates to high-value products. Here we leverage the proclivity by directing the C-C bond formation in a catalytic arylation pathway using lignin-derived phenols with high nucleophilicity. The selectively condensed lignin, isolated in near-quantitative yields while preserving its prominent cleavable ß-ether units, can be unlocked in a tandem catalytic process involving aryl migration and transfer hydrogenation. Lignin in wood is thereby converted to benign bisphenols (34-48 wt%) that represent performance-advantaged replacements for their fossil-based counterparts. Delignified pulp from cellulose and xylose from xylan are co-produced for textile fibres and renewable chemicals. This condensation-driven strategy represents a key advancement complementary to other promising monophenol-oriented approaches targeting valuable platform chemicals and materials, thereby contributing to holistic biomass valorization.

Copper-catalysed dehydrogenation or lactonization of C(sp3)-H bonds.

Cytochrome P450 enzymes are known to catalyse bimodal oxidation of aliphatic acids via radical intermediates, which partition between pathways of hydroxylation and desaturation1,2. Developing analogous catalytic systems for remote C-H functionalization remains a significant challenge3-5. Here, we report the development of Cu(I)-catalysed bimodal dehydrogenation/lactonization reactions of synthetically common N-methoxyamides through radical abstractions of the γ-aliphatic C-H bonds. The feasibility of switching from dehydrogenation to lactonization is also demonstrated by altering reaction conditions. The use of a readily available amide as both radical precursor and internal oxidant allows for the development of redox-neutral C-H functionalization reactions with methanol as the sole side product. These C-H functionalization reactions using a Cu(I) catalyst with loading as low as 0.5 mol.% is applied to the diversification of a wide range of aliphatic acids including drug molecules and natural products. The exceptional compatibility of this catalytic system with a wide range of oxidatively sensitive functionality demonstrates the unique advantage of using a simple amide substrate as a mild internal oxidant.

Structural insights into caspase ADPR deacylization catalyzed by a bacterial effector and host calmodulin.

Programmed cell death and caspase proteins play a pivotal role in host innate immune response combating pathogen infections. Blocking cell death is employed by many bacterial pathogens as a universal virulence strategy. CopC family type III effectors, including CopC from an environmental pathogen Chromobacterium violaceum, utilize calmodulin (CaM) as a co-factor to inactivate caspases by arginine ADPR deacylization. However, the molecular basis of the catalytic and substrate/co-factor binding mechanism is unknown. Here, we determine successive cryo-EM structures of CaM-CopC-caspase-3 ternary complex in pre-reaction, transition, and post-reaction states, which elucidate a multistep enzymatic mechanism of CopC-catalyzed ADPR deacylization. Moreover, we capture a snapshot of the detachment of modified caspase-3 from CopC. These structural insights are validated by mutagenesis analyses of CopC-mediated ADPR deacylization in vitro and animal infection in vivo. Our study offers a structural framework for understanding the molecular basis of arginine ADPR deacylization catalyzed by the CopC family.

Structural basis of K63-ubiquitin chain formation by the Gordon-Holmes syndrome RBR E3 ubiquitin ligase RNF216.

An increasing number of genetic diseases are linked to deregulation of E3 ubiquitin ligases. Loss-of-function mutations in the RING-between-RING (RBR) family E3 ligase RNF216 (TRIAD3) cause Gordon-Holmes syndrome (GHS) and related neurodegenerative diseases. Functionally, RNF216 assembles K63-linked ubiquitin chains and has been implicated in regulation of innate immunity signaling pathways and synaptic plasticity. Here, we report crystal structures of key RNF216 reaction states including RNF216 in complex with ubiquitin and its reaction product, K63 di-ubiquitin. Our data provide a molecular explanation for chain-type specificity and reveal the molecular basis for disruption of RNF216 function by pathogenic GHS mutations. Furthermore, we demonstrate how RNF216 activity and chain-type specificity are regulated by phosphorylation and that RNF216 is allosterically activated by K63-linked di-ubiquitin. These molecular insights expand our understanding of RNF216 function and its role in disease and further define the mechanistic diversity of the RBR E3 ligase family.

RNA helicase proteins as chaperones and remodelers.

Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are the promotion of rearrangements of structured RNAs and the remodeling of ribonucleoprotein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. Although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA, and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms.

Allosteric communication in the dynein motor domain.

Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.

GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding.

The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (ßα)8 TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here, we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry. During spontaneous folding, all elements of the DapA TIM barrel acquire structure simultaneously in a process associated with a long search time. In contrast, GroEL/ES accelerates folding more than 30-fold by catalyzing segmental structure formation in the TIM barrel. Segmental structure formation is also observed during the fast spontaneous folding of a structural homolog of DapA from a bacterium that lacks GroEL/ES. Thus, chaperonin independence correlates with folding properties otherwise enforced by protein confinement in the GroEL/ES cage. We suggest that folding catalysis by GroEL/ES is required by a set of proteins to reach native state at a biologically relevant timescale, avoiding aggregation or degradation.

Copper-catalysed enantioconvergent alkylation of oxygen nucleophiles.

Carbon-oxygen bonds are commonplace in organic molecules, including chiral bioactive compounds; therefore, the development of methods for their construction with simultaneous control of stereoselectivity is an important objective in synthesis. The Williamson ether synthesis, first reported in 18501, is the most widely used approach to the alkylation of an oxygen nucleophile, but it has significant limitations (scope and stereochemistry) owing to its reaction mechanism (SN2 pathway). Transition-metal catalysis of the coupling of an oxygen nucleophile with an alkyl electrophile has the potential to address these limitations, but progress so far has been limited2-7, especially with regard to controlling enantioselectivity. Here we establish that a readily available copper catalyst can achieve an array of enantioconvergent substitution reactions of α-haloamides, a useful family of electrophiles, by oxygen nucleophiles; the reaction proceeds under mild conditions in the presence of a wide variety of functional groups. The catalyst is uniquely effective in being able to achieve enantioconvergent alkylations of not only oxygen nucleophiles but also nitrogen nucleophiles, giving support for the potential of transition-metal catalysts to provide a solution to the pivotal challenge of achieving enantioselective alkylations of heteroatom nucleophiles.

Enantioconvergent Cu-catalysed N-alkylation of aliphatic amines.

Chiral amines are commonly used in the pharmaceutical and agrochemical industries1. The strong demand for unnatural chiral amines has driven the development of catalytic asymmetric methods1,2. Although the N-alkylation of aliphatic amines with alkyl halides has been widely adopted for over 100 years, catalyst poisoning and unfettered reactivity have been preventing the development of a catalyst-controlled enantioselective version3-5. Here we report the use of chiral tridentate anionic ligands to enable the copper-catalysed chemoselective and enantioconvergent N-alkylation of aliphatic amines with α-carbonyl alkyl chlorides. This method can directly convert feedstock chemicals, including ammonia and pharmaceutically relevant amines, into unnatural chiral α-amino amides under mild and robust conditions. Excellent enantioselectivity and functional-group tolerance were observed. The power of the method is demonstrated in a number of complex settings, including late-stage functionalization and in the expedited synthesis of diverse amine drug molecules. The current method indicates that multidentate anionic ligands are a general solution for overcoming transition-metal-catalyst poisoning.

Catalytic asymmetric synthesis of cannabinoids and menthol from neral.

The selective conversion of natural or synthetic neral to (1R,6S)-trans-isopiperitenol would enable and expedite sustainable routes to menthol1,2 and cannabinoids3-5. However, this reaction has been considered impossible because its product is more reactive to the required acid catalysts than its starting material, resulting in several side products6-9. We now show that an unsymmetric, strong and confined chiral acid, a highly fluorinated imino-imidodiphosphate, catalyses this process with excellent efficiency and selectivity. Expanding the method to other α,ß-unsaturated aldehydes could enable access to new cannabinoids and menthol derivatives not readily accessible previously. Mechanistic studies suggest that the confined catalyst accomplishes this reaction by binding the product in an unreactive conformation, thereby preventing its decomposition. We also show how (1R,6S)-trans-isopiperitenol can be readily converted to pharmaceutically useful cannabinoids and menthol, each in the shortest and most atom-economic routes so far.

Structural and mechanistic basis for protein glutamylation by the kinase fold.

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.