High-throughput reprogramming of an NRPS condensation domain.

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer.

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Cyanophycin and its biosynthesis: not hot but very cool.

Covering: 1878 to early 2023Cyanophycin is a biopolymer consisting of a poly-aspartate backbone with arginines linked to each Asp sidechain through isopeptide bonds. Cyanophycin is made by cyanophycin synthetase 1 or 2 through ATP-dependent polymerization of Asp and Arg, or ß-Asp-Arg, respectively. It is degraded into dipeptides by exo-cyanophycinases, and these dipeptides are hydrolyzed into free amino acids by general or dedicated isodipeptidase enzymes. When synthesized, chains of cyanophycin coalesce into large, inert, membrane-less granules. Although discovered in cyanobacteria, cyanophycin is made by species throughout the bacterial kingdom, and cyanophycin metabolism provides advantages for toxic bloom forming algae and some human pathogens. Some bacteria have developed dedicated schemes for cyanophycin accumulation and use, which include fine temporal and spatial regulation. Cyanophycin has also been heterologously produced in a variety of host organisms to a remarkable level, over 50% of the host's dry mass, and has potential for a variety of green industrial applications. In this review, we summarize the progression of cyanophycin research, with an emphasis on recent structural studies of enzymes in the cyanophycin biosynthetic pathway. These include several unexpected revelations that show cyanophycin synthetase to be a very cool, multi-functional macromolecular machine.

Bioinformatics of cyanophycin metabolism genes and characterization of promiscuous isoaspartyl dipeptidases that catalyze the final step of cyanophycin degradation.

Cyanophycin is a bacterial biopolymer used for storage of fixed nitrogen. It is composed of a backbone of L-aspartate residues with L-arginines attached to each of their side chains. Cyanophycin is produced by cyanophycin synthetase 1 (CphA1) using Arg, Asp and ATP, and is degraded in two steps. First, cyanophycinase breaks down the backbone peptide bonds, releasing ß-Asp-Arg dipeptides. Then, these dipeptides are broken down into free Asp and Arg by enzymes with isoaspartyl dipeptidase activity. Two bacterial enzymes are known to possess promiscuous isoaspartyl dipeptidase activity: isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA). We performed a bioinformatic analysis to investigate whether genes for cyanophycin metabolism enzymes cluster together or are spread around the microbial genomes. Many genomes showed incomplete contingents of known cyanophycin metabolizing genes, with different patterns in various bacterial clades. Cyanophycin synthetase and cyanophycinase are usually clustered together when recognizable genes for each are found within a genome. Cyanophycinase and isoaspartyl dipeptidase genes typically cluster within genomes lacking cphA1. About one-third of genomes with genes for CphA1, cyanophycinase and IaaA show these genes clustered together, while the proportion is around one-sixth for CphA1, cyanophycinase and IadA. We used X-ray crystallography and biochemical studies to characterize an IadA and an IaaA from two such clusters, in Leucothrix mucor and Roseivivax halodurans, respectively. The enzymes retained their promiscuous nature, showing that being associated with cyanophycin-related genes did not make them specific for ß-Asp-Arg dipeptides derived from cyanophycin degradation.

Structure and function of a hexameric cyanophycin synthetase 2.

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide ß-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.

Exploring the targeting spectrum of rocaglates among eIF4A homologs.

Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates-two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3-a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA transcripts. We find that rocaglates can also interact with eIF4A3 to induce RNA clamping. Rocaglates also inhibit EJC-dependent NMD in mammalian cells, but this does not appear to be due to induced eIF4A3-RNA clamping, but rather a secondary consequence of translation inhibition incurred by clamping eIF4A1 and eIF4A2 to mRNA.

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin.

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.

Evolution of naturally arising SARS-CoV-2 defective interfering particles.

Defective interfering (DI) particles arise during virus propagation, are conditional on parental virus for replication and packaging, and interfere with viral expansion. There is much interest in developing DIs as anti-viral agents. Here we characterize DI particles that arose following serial passaging of SARS-CoV-2 at high multiplicity of infection. The prominent DIs identified have lost ~84% of the SARS-CoV-2 genome and are capable of attenuating parental viral titers. Synthetic variants of the DI genomes also interfere with infection and can be used as conditional, gene delivery vehicles. In addition, the DI genomes encode an Nsp1-10 fusion protein capable of attenuating viral replication. These results identify naturally selected defective viral genomes that emerged and stably propagated in the presence of parental virus.

The structure of cyanophycinase in complex with a cyanophycin degradation intermediate.

BACKGROUND: Cyanophycinases are serine protease family enzymes which are required for the metabolism of cyanophycin, the natural polymer multi-L-arginyl-poly(L-aspartic acid). Cyanophycinases degrade cyanophycin to ß-Asp-Arg dipeptides, which enables use of this important store of fixed nitrogen. METHODS: We used genetic code expansion to incorporate diaminopropionic acid into cyanophycinase in place of the active site serine, and determined a high-resolution structure of the covalent acyl-enzyme intermediate resulting from attack of cyanophycinase on a short cyanophycin segment. RESULTS: The structure indicates that cyanophycin dipeptide residues P1 and P1' bind shallow pockets adjacent to the catalytic residues. We observe many cyanophycinase - P1 dipeptide interactions in the co-complex structure. Calorimetry measurements show that at least two cyanophycin dipeptides are needed for high affinity binding to cyanophycinase. We also characterized a putative cyanophycinase which we found to be structurally very similar but that shows no activity and could not be activated by mutation of its active site. GENERAL SIGNIFICANCE: Despite its peptidic structure, cyanophycin is resistant to degradation by peptidases and other proteases. Our results help show how cyanophycinase can specifically bind and degrade this important polymer.

A cryptic third active site in cyanophycin synthetase creates primers for polymerization.

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.

Structure and Function of a Dehydrating Condensation Domain in Nonribosomal Peptide Biosynthesis.

Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (CmodAA). We also obtain the crystal structure of a CmodAA domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-CmodAA modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-CmodAA identify several key residues for activity, including four residues that are mostly conserved in the CmodAA subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for CmodAA domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of CmodAA domains. Understanding CmodAA domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.

Structure and Function of the ß-Asp-Arg Polymerase Cyanophycin Synthetase 2.

Cyanophycin is a biopolymer composed of long chains of ß-Asp-Arg. It is widespread in nature, being synthesized by many clades of bacteria, which use it as a cellular reservoir of nitrogen, carbon, and energy. Two enzymes are known to produce cyanophycin: cyanophycin synthetase 1 (CphA1), which builds cyanophycin from the amino acids Asp and Arg by alternating between two separate reactions for backbone extension and side chain modification, and cyanophycin synthetase 2 (CphA2), which polymerizes ß-Asp-Arg dipeptides. CphA2 is evolutionarily related to CphA1, but questions about CphA2's altered structure and function remain unresolved. Cyanophycin and related molecules have drawn interest as green biopolymers. Because it only has a single active site, CphA2 could be more useful than CphA1 for biotechnological applications seeking to produce modified cyanophycin. In this study, we report biochemical assays on nine cyanobacterial CphA2 enzymes and report the crystal structure of CphA2 from Gloeothece citriformis at 3.0 Å resolution. The structure reveals a homodimeric, three-domain architecture. One domain harbors the polymerization active site and the two other domains have structural roles. The structure and biochemical assays explain how CphA2 binds and polymerizes ß-Asp-Arg and highlights differences in in vitro oligomerization and activity between CphA2 enzymes. Using the structure and distinct activity profile as a guide, we introduced a single point mutation that converted Gloeothece citriformis CphA2 from a primer-dependent enzyme into a primer-independent enzyme.

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Design, synthesis and in vitro evaluation of novel SARS-CoV-2 3CLpro covalent inhibitors.

Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CLpro (main coronaviruses cysteine-protease) has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CLpro non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CLpro. We report herein our efforts in the design and synthesis of submicromolar covalent inhibitors when the enzymatic activity of the viral protease was used as a screening platform.

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline.

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.

The chaperone HSPB1 prepares protein aggregates for resolubilization by HSP70.

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.

Structures and function of the amino acid polymerase cyanophycin synthetase.

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the ß-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

Structural basis for plazomicin antibiotic action and resistance.

The approval of plazomicin broadened the clinical library of aminoglycosides available for use against emerging bacterial pathogens. Contrarily to other aminoglycosides, resistance to plazomicin is limited; still, instances of resistance have been reported in clinical settings. Here, we present structural insights into the mechanism of plazomicin action and the mechanisms of clinical resistance. The structural data reveal that plazomicin exclusively binds to the 16S ribosomal A site, where it likely interferes with the fidelity of mRNA translation. The unique extensions to the core aminoglycoside scaffold incorporated into the structure of plazomicin do not interfere with ribosome binding, which is analogously seen in the binding of this antibiotic to the AAC(2')-Ia resistance enzyme. The data provides a structural rationale for resistance conferred by drug acetylation and ribosome methylation, i.e., the two mechanisms of resistance observed clinically. Finally, the crystal structures of plazomicin in complex with both its target and the clinically relevant resistance factor provide a roadmap for next-generation drug development that aims to ameliorate the impact of antibiotic resistance.

The multifaceted eukaryotic cap structure.

The 5' cap structure is added onto RNA polymerase II transcripts soon after initiation of transcription and modulates several post-transcriptional regulatory events involved in RNA maturation. It is also required for stimulating translation initiation of many cytoplasmic mRNAs and serves to protect mRNAs from degradation. These functional properties of the cap are mediated by several cap binding proteins (CBPs) involved in nuclear and cytoplasmic gene expression steps. The role that CBPs play in gene regulation, as well as the biophysical nature by which they recognize the cap, is quite intricate. Differences in mechanisms of capping as well as nuances in cap recognition speak to the potential of targeting these processes for drug development. In this review, we focus on recent findings concerning the cap epitranscriptome, our understanding of cap binding by different CBPs, and explore therapeutic targeting of CBP-cap interaction. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > Capping and 5' End Modifications Translation > Translation Mechanisms.