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.

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.

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.

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.

Whi3 mnemon association with endoplasmic reticulum membranes confines the memory of deceptive courtship to the yeast mother cell.

Prion-like proteins are involved in many aspects of cellular physiology, including cellular memory. In response to deceptive courtship, budding yeast escapes pheromone-induced cell-cycle arrest through the coalescence of the G1/S inhibitor Whi3 into a dominant, inactive super-assembly. Whi3 is a mnemon (Whi3mnem), a protein that conformational change maintains as a trait in the mother cell but is not inherited by the daughter cells. How the maintenance and asymmetric inheritance of Whi3mnem are achieved is unknown. Here, we report that Whi3mnem is closely associated with endoplasmic reticulum (ER) membranes and is retained in the mother cell by the lateral diffusion barriers present at the bud neck. Strikingly, barrier defects made Whi3mnem propagate in a mitotically stable, prion-like manner. The amyloid-forming glutamine-rich domain of Whi3 was required for both mnemon and prion-like behaviors. Thus, we propose that Whi3mnem is in a self-templating state, lending temporal maintenance of memory, whereas its association with the compartmentalized membranes of the ER prevents infectious propagation to the daughter cells. These results suggest that confined self-templating super-assembly is a powerful mechanism for the long-term encoding of information in a spatially defined manner. Yeast courtship may provide insights on how individual synapses become potentiated in neuronal memory.

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.

Cell Penetration, Herbicidal Activity, and in-vivo-Toxicity of Oligo-Arginine Derivatives and of Novel Guanidinium-Rich Compounds Derived from the Biopolymer Cyanophycin.

Oligo-arginines are thoroughly studied cell-penetrating peptides (CPPs, Figures 1 and 2). Previous in-vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti-malaria drug) have shown a 40-fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in-vitro and in-vivo activity was demonstrated with various cell-penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 µmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly-aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H-ßAsp-αArg-OH (H-Adp-OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8-derivatives (Figure 5). Intravenouse injection of H-Adp8-NH2 into the tail vein of mice with doses as high as 45 µmol/kg causes no symptoms whatsoever (Table 3), but H-Adp8-NH2 is not cell penetrating (HEK293 and MCF-7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM-(Adp(OMe))8-NH2 and FAM-(Adp(NMe2))8-NH2 with ester and amide groups in the side chains exhibit mediocre to high cell-wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8).

Surface-Engineered Cationic Nanocrystals Stable in Biological Buffers and High Ionic Strength Solutions.

Progress in colloidal synthesis in the last two decades has enabled high-quality semiconductor, plasmonic, and magnetic nanocrystals (NCs). As synthesized, these NCs are usually capped with long-chain apolar ligands. Postsynthetic surface functionalization is required for rendering such NCs colloidally stable in polar media such as water. However, unlike small anionic molecules and polymeric coatings, producing positively charged stable NCs, especially at high ionic strengths, has remained challenging. Here, we present a general approach to achieve aqueously stable cationic NCs using a set of small (<2.5 nm long) positively charged ligands. The applicability of this method is demonstrated for a variety of materials including semiconductor CdSe/CdS core/shell NCs, magnetic Fe@Fe3O4, Fe3O4, and FePt NCs, and three different classes of plasmonic Au NCs including large nanorods. The obtained cationic NCs typically have zeta potential values ranging from +30 to +60 mV and retain colloidal stability for days to months, depending on NC/ligand pair, in several biological buffers at elevated pH and in concentrated salt solutions. This allowed us to demonstrate site-specific staining of cellular structures using fluorescent cationic NCs with several different surface chemistries. Furthermore, colloidal stability of the obtained NCs in the presence of other charged species allowed the assembly of cationic and anionic counterparts driven primarily by electrostatic attraction. With this approach, we prepare highly uniform 3D and 2D binary mixtures of NCs through induced homogeneous aggregation and alternating-charge layer-by-layer deposition, respectively. Such binary mixtures may provide a new route in the engineering of nanocrystalline solids for electronics, thermoelectrics, and photovoltaics.

Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable Self-Assembly of Distinct Amyloid Structures.

Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human ß2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids.

Chiral recognition in amyloid fiber growth.

Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human ß2-microglobulin (ß2m) and the 99-residue full-length protein. For the dimeric [20-41]ß2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length ß2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-ß2m amyloids. Furthermore, the full-length d-enantiomer of ß2m was found to be an efficient inhibitor of l-ß2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.