Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat). Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln). Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex

Interactions of the human LIP5 regulatory protein with endosomal sorting complexes required for transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic "leucine collar" that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate

Production of Site-Specific Antibody–Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System

Antibody–drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide–alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in <i>in vitro</i> cell cytotoxicity assays

Essential Oils from Aromatic and Medicinal Plants as Effective Weapons Against Mosquito Vectors of Public Health Importance

The fight against mosquito-borne diseases has recently seen the failure of control programmes based on synthetic chemical treatments to combat larvae and adults of mosquito vectors. This has led to several problems linked to residual substances causing a detrimental impact on environment and human health and to the development of resistance in mosquitoes. In this scenario, new eco-friendly and alternative strategies for the management of mosquito-borne diseases come from the use of plant essential oils (EOs). These are complex mixtures of small, volatile and lipophilic compounds, mostly belonging to monoterpenoids, sesquiterpenoids and phenylpropanoids, produced by aromatic plants belonging to several botanical families such as Apiaceae, Asteraceae, Geraniaceae, Lamiaceae, Lauraceae, Myrtaceae, Poaceae, Rutaceae, Verbenaceae and Zingiberaceae. An important ecological role played by EOs is defending plants from several enemies such as bacterial and fungal pathogens, viruses, insects and parasites. EOs represent ideal candidate ingredients to be incorporated in insecticidal formulations since scientific evidences have documented their efficacy against larvae and adults of several mosquitoes (e.g. Anopheles, Aedes and Culex) even at low doses (&lt;50 ppm), the multiple mode of action and wide spectrum of efficacy, the low toxicity on nontarget organisms and environment and the unlikely capacity to induce insect resistance. In this chapter, we gave an overview of the most important EOs obtained from commercially important botanical families with documented efficacy against mosquito vectors. Particular attention has been paid to highlight their strengths and weakness and the future challenges leading to the replacement of conventional insecticides by agrochemical companies