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Structure-activity studies with site-specifically modified apoptin in living cells demonstrate that this protein's toxicity is mediated by phosphorylation and downstream interactions with the cytoskeleton.

The site-selective modification of peptides and proteins facilitates the preparation of targeted therapeutic agents and tools to interrogate fundamental biochemistry. Among the numerous bioconjugation techniques developed to install groups of interest, those that generate C(sp3)-C(sp3) bonds are significantly underrepresented despite affording proteolytically stable, biogenic linkages. Herein we describe a visible-light-mediated reaction that enables the site-selective modification of peptides and proteins via desulfurative C(sp3)-C(sp3) bond formation. The reaction is rapid and high yielding in peptide systems, with comparable translation to proteins. Using this chemistry, we demonstrate the installation of a range of moieties into model systems and successfully integrate an effective PTM-mimic into a recombinantly expressed histone.

Apoptin is a small viral protein capable of inducing cell death selectively in cancer cells. Despite its potential as an anticancer agent, relatively little is known about its mechanism of toxicity and cancer-selectivity. Previous experiments suggest that cancer-selective phosphorylation modulates Apoptin toxicity, although a lack of chemical tools has hampered the dissection of underlying mechanisms. Here, we describe structure-function studies with site-specifically phosphorylated Apoptin (Apoptin-T108ph) in living cells which revealed that Thr108 phosphorylation is the selectivity switch for Apoptin toxicity. Mechanistic investigations link T108ph to actin binding, cytoskeletal disruption and downstream inhibition of anoikis-resistance as well as cancer cell invasion. These results establish Apoptin as a protein pro-drug, selectively activated in cancer cells by phosphorylation, which disrupts the cytoskeleton and promotes cell death. We anticipate that this mechanism provides a framework for the design of next generation anticancer proteins with enhanced selectivity and potency.

The tumor suppressor protein p53 is a master regulator of cell fate. The activity of p53 is controlled by a plethora of posttranslational modifications (PTMs). However, despite extensive research, the mechanisms of this regulation are still poorly understood due to a paucity of biochemical studies with p53 carrying defined PTMs. Here, we report a protein semi-synthesis approach to access site-specifically modified p53. We synthesized a set of chemically homogeneous full-length p53 carrying one (Ser20ph and Ser15ph) or two (Ser15,20ph) naturally occurring, damage-associated phosphoryl marks. Refolding and biochemical characterization of semisynthetic p53 variants confirmed their structural and functional integrity. Furthermore, we show that phosphorylation within the N-terminal domain directly enhances p300-dependent acetylation approximately twofold, consistent with the role of these marks in p53 activation. Given that the p53 N-terminus is a hotspot for PTMs, we believe that our approach will contribute greatly to a mechanistic understanding of how p53 is controlled by PTMs., ‘Designer’ p53: semi-synthesis of the tumor suppressor protein p53 via native chemical ligation enables in vitro structure–activity studies to reveal how this master regulator of cell fate is itself regulated by phosphorylation.

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their β-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a β-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Polycomb Repressive Complex 2 (PRC2) catalyzes mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27me1-3) to control expression of genes important for differentiation and maintenance of cell identity. PRC2 activity is regulated by a number of different inputs, including allosteric activation by its product, H3K27me3. This positive feedback loop is thought to be important for the establishment of large domains of condensed heterochromatin. In addition to other chromatin modifications, ancillary subunits of PRC2, foremost JARID2, affect the rate of H3K27 methylation. Many gaps remain in our understanding of how PRC2 integrates these various signals to determine where and when to deposit H3K27 methyl marks. In this study, we utilize designer chromatin substrates to demonstrate that propagation of H3K27 methylation by the PRC2 core complex has geometrically defined preferences that are overridden by the presence of JARID2. Our studies also show that phosphorylation of JARID2 can partially regulate its ability to stimulate PRC2 activity. Collectively, these biochemical insights further our understanding of the mechanisms that govern PRC2 activity, and highlight a role for JARID2 in de novo deposition of H3K27me3-containing repressive domains.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 “senses” H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the k(cat) of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.

Specialized chromatin domains contribute to nuclear organization and regulation of gene expression. Gene-poor regions are di-and trimethylated at lysine 9 of histone H3 (H3K9me2 and H3K9me3) by the histone methyltransferase Suv39h1. This enzyme harnesses a positive feedback loop to spread H3K9me2 and H3K9me3 over extended heterochromatic regions. However, little is known about how feedback loops operate on complex biopolymers such as chromatin, in part because of the difficulty in obtaining suitable substrates. Here we describe the synthesis of multidomain 'designer chromatin' templates and their application to dissecting the regulation of human Suv39h1. We uncovered a two-step activation switch where H3K9me3 recognition and subsequent anchoring of the enzyme to chromatin allosterically promotes methylation activity and confirmed that this mechanism contributes to chromatin recognition in cells. We propose that this mechanism serves as a paradigm in chromatin biochemistry, as it enables highly dynamic sampling of chromatin state combined with targeted modification of desired genomic regions.

A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream from transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes and affects codon usage and amino acid composition in genes. Given that the AT-rich Tetrahymena genome is intrinsically unfavorable for nucleosome formation, we propose that these “seed” nucleosomes—together with trans-acting factors—may facilitate the establishment of nucleosome arrays within genes in vivo, while minimizing changes to the underlying coding sequences.

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native (i.e. cellular) chromatin, each nucleosome can differ from its neighbors through the localized installation of covalent modifications to both the genomic DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation and that, as a consequence, must employ compositionally defined chromatin substrates. Here, we introduce a chemical approach to the production of heterotypic ‘designer’ chromatin that can be used in such studies. Our method involves attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a Peptide Nucleic Acid (PNA) targeting compound. We apply this strategy to dissect the role of chromatin context on both the activation and inhibition of the histone methyltransferase, PRC2, which methylates Lys 27 of histone H3 (H3K27). Our studies show that PRC2 can be stimulated to produce de novo H3K27 methylation from a defined nucleation site. More generally, this technology promises to facilitate biochemical studies that require the use of heterotypic chromatin substrates.

Synthetic protein chemistry enables precise control of the chemical makeup of proteins and facilitates the incorporation of new functionalities and connectivities. This chapter covers the principles of protein synthesis, by solid-phase peptide synthesis one amino acid at a time and through expedient fragment assembly via convergent methods. General applications of synthetic proteins, as well as a series of case studies that demonstrate the utility and versatility of synthetic protein chemistry for solving biological problems are discussed.

Post-translational modifications (PTMs) dramatically enhance the capabilities of proteins. They introduce new functionalities and dynamically control protein activity by modulating intra- and intermolecular interactions. Traditionally, PTMs have been considered as reversible attachments to nucleophilic functional groups on amino acid side chains, whereas the polypeptide backbone is often thought to be inert. This paradigm is shifting as chemically and functionally diverse alterations of the protein backbone are discovered. Importantly, backbone PTMs can control protein structure and function just as side chain modifications do and operate through unique mechanisms to achieve these features. In this Perspective, I outline the various types of protein backbone modifications discovered so far and highlight their contributions to biology as well as the challenges in studying this versatile yet poorly characterized class of PTMs.

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo1. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features2. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch3 is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface4,5,6,7,8,9.

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

Cell death is a tightly regulated process which can be exploited in cancer treatment to drive the killing of the tumour. Several conventional cancer therapies including chemotherapeutic agents target pathways involved in cell death, yet they often fail due to the lack of selectivity they have for tumour cells over healthy cells. Over the past decade, research has demonstrated the existence of numerous proteins which have an intrinsic tumour-specific toxicity, several of which originate from viruses. These tumour-selective viral proteins, although from distinct backgrounds, have several similar and interesting properties. Though the mechanism(s) of action of these proteins are not fully understood, it is possible that they can manipulate several cell death modes in cancer exemplifying the intricate interplay between these pathways. This review will discuss our current knowledge on the topic and outstanding questions, as well as deliberate the potential for viral proteins to progress into the clinic as successful cancer therapeutics.

Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes containing foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones.

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub), a post-translational modification first discovered in 1980, plays a critical role in diverse nuclear processes including the regulation of transcription and DNA damage repair. Herein, we use a suite of protein chemistry methods to explore how H2B-Ub stimulates hDot1L-mediated methylation of histone H3 on lysine 79 (H3K79me). By using semisynthetic ‘designer' chromatin containing H2B-Ub bearing a site-specifically installed photocrosslinker, here we report an interaction between a functional hotspot on ubiquitin and the N-terminus of histone H2A. Our biochemical studies indicate that this interaction is required for stimulation of hDot1L activity and leads to a repositioning of hDot1L on the nucleosomal surface, which likely places the active site of the enzyme proximal to H3K79. Collectively, our data converge on a possible mechanism for hDot1L stimulation in which H2B-Ub physically ‘corrals' the enzyme into a productive binding orientation., The ubiquitylation of histone H2B on lysine 120 is an important modification with roles in a diverse range of nuclear processes. Here, the authors use 'designer' chromatin to show that H2B-ub orients hDot1L into the correct position for activation.

Split proteins are versatile tools for detecting protein–protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein–protein interactions.

Split proteins are versatile tools for detecting protein–protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein–protein interactions.

Current strategies for creating enzyme-like catalysts range from rational[1] and computational design[2] to evolutionary searches of large molecular libraries.[3] Sequence-specific polymers are particularly attractive starting points for these efforts because of their ability to adopt three-dimensional structures that preorganize functional groups for catalysis. Although natural enzymes are constructed from α-amino acids, many other backbone structures can give rise to well-defined secondary and tertiary structures. Such non-natural oligomers, often referred to as “foldamers”, have the potential to display properties akin to those of proteins.[4–8]

A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within.

In eukaryotic cells, inheritable information is stored in a nucleoprotein complex referred to as chromatin.1 This genome architecture serves two key purposes. On the one hand, wrapping DNA (approximately 145–147 basepairs) twice around a spool composed of two copies each of the highly basic core histones H2A, H2B, H3, and H4 leads to compaction of DNA strands (Figure ​(Figure1a,b).1a,b). These assemblies are called nucleosomes. Contacts between individual nucleosomes are often mediated by cationic tails at the N- and C-termini of all histone proteins that protrude from the core and further tighten the chromatin fiber (Figure ​(Figure1c).1c). Additional packing is achieved through attachment of histone H1 to the DNA that links neighboring nucleosomes or by nonhistone proteins that are able to bridge units within or between chromatin fibers.2 The second pivotal function of storing genetic information as a DNA–protein complex is the additional layer of regulation that this feature provides.3−5 For instance, the very presence of histones on DNA sequences can occlude access to these sites by transcription factors and other DNA binding proteins.6 Thus, nucleosome positioning, shaped in part by DNA sequence preferences and shifted by ATP-powered molecular motors (referred to as chromatin remodelers), directly affects chromatin transactions.7 Beyond their location, the biochemical makeup of nucleosomes provides further opportunity for regulation. Canonical histones can be replaced with closely resembling variants, and all histones are dynamically decorated with post-translational modifications (PTMs). These biochemical marks can be as small as just a few atoms, such as methyl (Lys, Arg, Gln), acetyl (Lys), or phosphoryl groups (Ser, Thr), or as large as an entire protein in the case of ubiquitin or SUMO. Upon attachment by dedicated transferase enzymes, PTMs can directly alter the biophysical properties of the target protein, provide a docking site for specific interaction partners, interfere with binding events of other factors, or act through a combination of these mechanisms. In this way, signaling through histone PTMs serves to orchestrate chromatin-templated processes, including fine-tuning transcriptional outputs. Remarkably, transcriptional states can be inherited through cell division cycles, thus providing a mode of epigenetic memory.8,9 Not surprisingly, misregulation of the inputs and outputs of chromatin signaling occurs in many diseases, especially cancer.10−13 Figure 1 Chromatin architecture in eukaryotic cells. (a) Structure of a mononucleosome. DNA (gray) is wrapped around two copies each of H2A (orange), H2B (red), H3 (blue), and H4 (green); pdb code: 1kx5. (b) Electrostatic surface rendering of a histone octamer. ...

The histone methyltransferase PRC2 plays a central role in genomic stability and cellular development. Consequently, its misregulation has been implicated in several cancers. Recent work has shown that a histone H3 mutant, where the PRC2 substrate residue Lys27 is replaced by methionine, is also associated with cancer phenotypes and functions as an inhibitor of PRC2. Here we investigate the mechanism of this PRC2 inhibition through kinetic studies and photo-cross-linking. Efficient inhibition is dependent on (1) hydrophobic lysine isosteres blocking the active site, (2) proximal residues, and (3) the H3 tail forming extensive contacts with the EZH2 subunit of PRC2. We further show that naturally occurring post-translational modifications of the same H3 tail, both proximal and distal to K27M, can greatly diminish the inhibition of PRC2. These results suggest that this potent gain of function mutation may be “detoxified” by modulating alternate chromatin modification pathways.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function., PLoS Genetics, 9 (1), ISSN:1553-7390, ISSN:1553-7404