Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio-Functionalization of PET.

We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.

Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid.

Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates1. These enzymes include serine hydrolases2,3 (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery4,5. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours6. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight7-10, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code11. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase12, we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.

Mitotic phosphorylation regulates Hsp72 spindle localization by uncoupling ATP binding from substrate release.

Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21.

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKß and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.

Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.

Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing.

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel positive selections combined with deep sequencing and statistical analysis and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNACUA pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure determination and synthetic protein kinase activation.

Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.

Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog.

Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery.

Aldehyde drugs are gaining increasing research interest, considering that aldehyde dehydrogenases overexpression is characteristic of cancer stem cells. Here, we describe the traceless site-specific coupling of a novel potent drug, containing an aldehyde moiety, to recombinant antibodies, which were engineered to display a cysteine residue at their N-terminus, or a 1,2-aminothiol at their C-terminus. The resulting chemically defined antibody-drug conjugates represent the first example in which a thiazolidine linkage is used for the targeted delivery and release of cytotoxic agents.