Systematic Variation of Both the Aromatic Cage and Dialkyllysine via GCE-SAR Reveal Mechanistic Insights in CBX5 Reader Protein Binding.

Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein-methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure-activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation-π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends.

Design and Evaluation of Artificial Hybrid Photoredox Biocatalysts.

A pair of 9-mesityl-10-phenyl acridinium (Mes-Acr+ ) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes-Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes-Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes-Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones.

Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from Drosophila melanogaster and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader-H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers (eReaders) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes in vitro, demonstrating the utility of eReaders to complement antibodies in epigenetics research.

Thermodynamic consequences of Tyr to Trp mutations in the cation-π-mediated binding of trimethyllysine by the HP1 chromodomain.

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains.

Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug.

Polypeptides are promising carriers for chemotherapeutics: they have minimal toxicity, can be recombinantly synthesized with precise control over molecular weight, and enhance drug pharmacokinetics as self-assembled nanoparticles. Polypeptide-based systems also provide the ability to achieve active targeting with genetically encoded targeting ligands. While passive targeting promotes accumulation of nanocarriers in solid tumors, active targeting provides an additional layer of tunable control and widens the therapeutic window. However, fusion of most targeting proteins to polypeptide carriers exposes the limitations of this approach: the residues that are used for drug attachment are also promiscuously distributed on protein surfaces. We present here a universal methodology to solve this problem by the site-specific attachment of extrinsic moieties to polypeptide drug delivery systems without cross-reactivity to fused targeting domains. We incorporate an unnatural amino acid, p-acetylphenylalanine, to provide a biorthogonal ketone for attachment of doxorubicin in the presence of reactive amino acids in a nanobody-targeted, elastin-like polypeptide nanoparticle. These nanoparticles exhibit significantly greater cytotoxicity than nontargeted controls in multiple cancer cell lines.

Osmolytes and Protein-Protein Interactions.

Cells survive fluctuations in osmolality by accumulating and depleting highly soluble, usually neutral, small organic compounds. Natural selection has converged on a small set of such molecules, called osmolytes. The biophysical characterization of osmolytes, with respect to proteins, has centered on tertiary structure stability. Data about their effect on protein assemblies, whose formation is driven by surface interactions, is lacking. Here, we investigate the effects of osmolytes and related molecules on the stabilities of a protein and a protein complex. The results demonstrate that osmolytes are not differentiated from other cosolutes by their stabilizing influences on protein tertiary structure but by their compatibility with the interactions between protein surfaces that organize the cellular interior.

Enhancing cytochrome P450-mediated non-natural cyclopropanation by mutation of a conserved second-shell residue.

Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h . Here, we demonstrate that mutation of a ubiquitously conserved second-shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first-shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non-natural catalysis in 4 diverse P450 scaffolds.

Photo-Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano- to Microgels of Intrinsically Disordered Polypeptides.

Hydrogel particles are versatile materials that provide exquisite, tunable control over the sequestration and delivery of materials in pharmaceutics, tissue engineering, and photonics. The favorable properties of hydrogel particles depend largely on their size, and particles ranging from nanometers to micrometers are used in different applications. Previous studies have only successfully fabricated these particles in one specific size regime and required a variety of materials and fabrication methods. A simple yet powerful system is developed to easily tune the size of polypeptide-based, thermoresponsive hydrogel particles, from the nano- to microscale, using a single starting material. Particle size is controlled by the self-assembly and unique phase transition behavior of elastin-like polypeptides in bulk and within microfluidic-generated droplets. These particles are then stabilized through ultraviolet irradiation of a photo-crosslinkable unnatural amino acid (UAA) cotranslationally incorporated into the parent polypeptide. The thermoresponsive property of these particles provides an active mechanism for actuation and a dynamic responsive to the environment. This work represents a fundamental advance in the generation of crosslinked biomaterials, especially in the form of soft matter colloids, and is one of the first demonstrations of successful use of UAAs in generating a novel material.

Investigation of Trimethyllysine Binding by the HP1 Chromodomain via Unnatural Amino Acid Mutagenesis.

Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

Orthogonal Expression of an Artificial Metalloenzyme for Abiotic Catalysis.

A cytochrome P450 was engineered to selectively incorporate Ir(Me)-deuteroporphyrin IX (Ir(Me)-DPIX), in lieu of heme, in bacterial cells. Cofactor selectivity was altered by introducing mutations within the heme-binding pocket to discriminate the deuteroporphyrin macrocycle, in combination with mutations to the P450 axial cysteine to accommodate a pendant methyl group on the Ir(Me) center. This artificial metalloenzyme was investigated for activity in non-native metallocarbenoid-mediated olefin cyclopropanation reactions and showed enhanced activity for aliphatic and electron-deficient olefins when compared to the native heme enzyme. This work provides a general strategy to augment the chemical functionality of heme enzymes in cells with application towards abiotic catalysis.

P450-Mediated Non-natural Cyclopropanation of Dehydroalanine-Containing Thiopeptides.

Thiopeptides are a growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Many biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous and can accept a wide range of peptide substrates with different amino acid sequences; thus, these enzymes have been used as tools to generate new natural product derivatives. Here, we explore an alternative route to molecular complexity by engineering thiopeptide tailoring enzymes to do new or non-native chemistry. We explore cytochrome P450 enzymes as biocatalysts for cyclopropanation of dehydroalanines, chemical motifs found widely in thiopeptides and other RiPP-based natural products. We find that P450TbtJ1 and P450TbtJ2 selectively cyclopropanate dehydroalanines in a number of complex thiopeptide-based substrates and convert them into 1-amino-2-cyclopropane carboxylic acids (ACCAs), which are important pharmacophores. This chemistry takes advantage of the innate affinity of these biosynthetic enzymes for their substrates and enables incorporation of new pharmacophores into thiopeptide architectures. This work also presents a strategy for diversification of natural products through rationally repurposing biosynthetic enzymes as non-natural biocatalysts.

Non-natural carbenoid and nitrenoid insertion reactions catalyzed by heme proteins.

Despite increasing interest in using enzymes as tools for synthesis, many reactions discovered through the creativity of synthetic chemists remain beyond the scope of biocatalysis. This vacancy in the field has compelled researchers to develop strategies to adapt protein scaffolds for new reactivity. Heme proteins have recently been shown to activate synthetic precursors to generate reactive metallocarbenoid and metallonitrenoid species that enable the biosynthetic construction of novel C-C, C-N, and other bonds using mechanisms not previously explored by Nature. By interrogating heme proteins with synthetic, non-natural reagents, scientists are merging the reaction space traditionally dominated by organocatalysis and transition metal catalysis with the mild reaction conditions, selectivity, and adaptability imparted by native protein scaffolds.

An Evolved Orthogonal Enzyme/Cofactor Pair.

We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.

Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation.

Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3 ) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers.

Tissue-specific proteome tagging: an orthogonal approach.

SORTing through the proteome: A versatile method has been developed for the incorporation of a novel 1,3-disubstituted cyclopropene amino acid into the proteome in response to a variety of sense codons and subsequent labeling. Incorporation can be confined to certain tissues by the selective expression of an orthogonal tRNA synthetase under cell-selective promoters.

A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo.

Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe(III)-to-Fe(II) reduction potential and substantially improves NAD(P)H-driven activity.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.