Susan E. Cellitti

A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

In vivo incorporation of unnatural amino acids to probe structure, dynamics and ligand binding in a large protein by Nuclear Magnetic Resonance spectroscopy

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.

Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824.

Summary Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.

D-Ornithine coopts pyrrolysine biosynthesis to make and insert pyrroline-carboxy-lysine

D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.

Efforts Toward the Direct Experimental Characterization of Enzyme Microenvironments: Tyrosine100 in Dihydrofolate Reductase†

State secrets: Site-specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occurring between Tyr100 and the charge that develops in the hydride-transfer transition state (see picture, NADP(+) purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code.

Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell–cell communication, viral entry and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in Escherichia coli through the use of an expanded genetic code that co-translationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY, and their use, along with a simple chemical synthesis of sulfotyrosine, are outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine sulfate is TAG. Co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in <1 week.

Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis.

Hydrogen exchange (HX) studies have provided critical insight into our understanding of protein folding, structure, and dynamics. More recently, hydrogen exchange mass spectrometry (HX-MS) has become a widely applicable tool for HX studies. The interpretation of the wealth of data generated by HX-MS experiments as well as other HX methods would greatly benefit from the availability of exchange predictions derived from structures or models for comparison with experiment. Most reported computational HX modeling studies have employed solvent-accessible-surface-area based metrics in attempts to interpret HX data on the basis of structures or models. In this study, a computational HX-MS prediction method based on classification of the amide hydrogen bonding modes mimicking the local unfolding model is demonstrated. Analysis of the NH bonding configurations from molecular dynamics (MD) simulation snapshots is used to determine partitioning over bonded and nonbonded NH states and is directly mapped into a protection factor (PF) using a logistics growth function. Predicted PFs are then used for calculating deuteration values of peptides and compared with experimental data. Hydrogen exchange MS data for fatty acid synthase thioesterase (FAS-TE) collected for a range of pHs and temperatures was used for detailed evaluation of the approach. High correlation between prediction and experiment for observable fragment peptides is observed in the FAS-TE and additional benchmarking systems that included various apo/holo proteins for which literature data were available. In addition, it is shown that HX modeling can improve experimental resolution through decomposition of in-exchange curves into rate classes, which correlate with prediction from MD. Successful rate class decompositions provide further evidence that the presented approach captures the underlying physical processes correctly at the single residue level. This assessment is further strengthened in a comparison of residue resolved protection factor predictions for staphylococcal nuclease with NMR data, which was also used to compare prediction performance with other algorithms described in the literature. The demonstrated transferable and scalable MD based HX prediction approach adds significantly to the available tools for HX-MS data interpretation based on available structures and models.

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.