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Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon-independent; the anticodon does not contact the enzyme. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a non-canonical amino acid.

Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C(pro)) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C(pro) make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C(pro) and analyzed its enzymatic activity. The crystal structure shows that EV71 3C(pro) has a typical chymotrypsin-like fold that is common in picornaviral 3C(pro). Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C(pro). We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C(pro) proteolytic activities. In summary, our work provides the first structural insight into EV71 3C(pro), including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C(pro).

SUMMARY Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant, suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin, and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.

EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C pro /rupintrivir complex, showing that although rupintrivir interacts with EV71 3C pro similarly to HRV 3C pro , the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C pro . Our structures reveal that EV71 3C pro possesses a surface-recessive S2 pocket that is not present in HRV 3C pro that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3Cpro , that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.

The central melanocortin system is critical in the regulation of appetite and body weight, and leptin exerts its anorexigenic actions partly by increasing hypothalamic proopiomelanocortin (POMC) expression. The POMC-derived peptide ␣-melanocytestimulating hormone (␣MSH) is a melanocortin 4 receptor agonist, and its potency in reducing energy intake is strongly increased by N-acetylation. The reason for the higher biological activity of N-acetylated ␣MSH (Act-␣MSH) compared with that of N-desacetylated ␣MSH (Des-␣MSH) is unclear, and regulation of acetylation by leptin has not been investigated. We show here that total hypothalamic ␣MSH levels are decreased in leptin-deficient ob͞ob mice and increased in leptin-treated ob͞ob and C57BL͞6J mice. The increase in total ␣MSH occurred as soon as 3 h after leptin injection and was entirely due to an increase in Act-␣MSH. Consistent with this observation, leptin rapidly induced the enzymatic activity of a N-acetyltransferase in the hypothalamus of mice. In 293T cells expressing the melanocortin 4 receptor, Act-␣MSH is far more potent than Des-␣MSH in stimulating cAMP accumulation, an effect caused by a dramatically increased stability of Act-␣MSH. Moreover, Des-␣MSH is rapidly degraded in the hypothalamus after intracerebroventricular injection in rats and was less potent in inhibiting energy intake. The results suggest that leptin activates a N-acetyltransferase in POMC neurons, leading to increased hypothalamic levels of Act-␣MSH. Due to its increased stability, this posttranslational modification of ␣MSH may play a critical role in leptin action via the central melanocortin pathway.T he adipocyte-derived hormone leptin is expressed in fat tissue and acts on the central nervous system to inform key regulatory centers about energy stores (1-3). Leptin receptors (ObRs) are highly expressed within the hypothalamus, including in proopiomelanocortin (POMC) neurons located in the arcuate nucleus (4, 5). POMC-derived neuropeptides, primarily ␣-melanocyte-stimulating hormone (␣MSH), activate the melanocortin 4 receptor (MC4R) and function downstream of leptin signaling to regulate energy balance, although other pathways are also important (6-10). Supporting the role of the melanocortin pathway in leptin action are data showing that central injection of synthetic melanocortin receptor antagonists inhibits the effect of leptin to reduce food intake (10). Furthermore, mutations in the pomc and mc4r genes result in severe obesity in mice (6, 11) and humans (7,12).Studies investigating the regulation of POMC neurons by leptin have focused mainly on gene expression analyses. It is known that fasting, which is a state of low serum leptin levels, results in reduced amounts of POMC mRNA levels. This reduction is also seen in the hypothalamus of the leptin-deficient ob͞ob mice (13-16). In addition, hypothalamic POMC mRNA is stimulated by the administration of recombinant leptin to rodents (16). However, few studies have examined regulation of hypothalamic POMC-derived peptides, such as ...

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