Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-ß-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 µM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Neutralization of matrix metalloproteinase-9 potentially enhances oncolytic efficacy of tanapox virus for melanoma therapy.

Matrix metalloproteinases (MMPs), which are involved in degradation of extracellular matrix, are critical regulators in tumor progression, metastasis and angiogenesis. Although induction of MMPs is frequently observed during the viral infection, the effect of MMPs on viral replication varies between viruses. MMP-9, for instance, is upregulated and promotes the replication of some viruses, such as herpes simplex virus, but inhibits the replication of others. Here, we report that infection with tanapox virus (TPV) promotes the expression of MMP-9 in the melanoma cells. In addition, we show that MMP-9 exerts an anti-viral effect on TPV replication and plays a protective role in TPV-infected melanoma cells in vitro. Moreover, the neutralization of MMP-9 in melanoma cells remarkably enhances the TPV infection and leads to a significant reduction in cell survival. In summary, this study contributes to understanding of the role played by MMP-9 in TPV infectivity and provides more insights for using TPV as cancer virotherapy in future studies. Since TPV has shown substantial oncolytic efficacy in promoting melanoma tumor regression in animal models, identifying mechanisms that suppress MMP-9 expression upon TPV infection can potentially improve its use as a melanoma virotherapy.

Identification of metabolites produced during the complete biodegradation of 1-butyl-3-methylimidazolium chloride by an enriched activated sludge microbial community.

Ionic liquids (ILs) are highly polar solvents with unique physicochemical properties that make them promising green alternatives to volatile organic solvents. Since ILs can be toxic to organisms, the development of methods to degrade ILs into harmless molecules prior to disposal is critical to enhancing their green properties. In this study, metabolites generated during the biodegradation of 1-butyl-3-methylimidazolium chloride (BMIMCl) by an enriched, activated sludge microbial community were investigated. Biodegradation of BMIM and the metabolic products released into the growth media were examined using 1H-NMR spectroscopy and mass spectrometry. To the best of our knowledge, this is the first reported complete primary catabolism of the biodegradation-resistant BMIMCl ionic liquid. The bacterial community responsible for degradation was analyzed using a 16S-rRNA amplicon approach. Although the community was diverse, Bacteroidetes was the predominant phylum. The study provides a greater insight into imidazolium-based IL biodegradability and a means to proactively prevent the ecotoxicity of the BMIM cation and its metabolites, by complete primary biodegradation of the cation and removal of most resulting metabolites, prior to release into aquatic waste streams.

Survival of the Fittest Nanojar: Stepwise Breakdown of Polydisperse Cu27-Cu31 Nanojar Mixtures into Monodisperse Cu27(CO3) and Cu31(SO4) Nanojars.

Nanojars are emerging as a class of anion sequestration agents of unparalleled efficiency. Dinegative oxoanions (e.g., carbonate, sulfate) template the formation of a series of homologous nanojars [Cu(OH)(pyrazolato)]n (n=27-31). Pyridine selectively transforms less stable, larger CO3(2-) nanojars (n=30, 31) into more stable, smaller ones (n=27, 29), but leaves all SO4(2-) nanojars (n=27-29, 31) intact. Ammonia, in turn, transforms all less stable nanojars into the most stable one and allows the isolation of pure [CO3(2-)⊂{Cu(OH)(pz)}27] and [SO4(2-)⊂{Cu(OH)(pz)}31]. A comprehensive picture of the solution and solid-state intricacies of nanojars was revealed by a combination of variable temperature NMR spectroscopy, tandem mass spectrometry, and X-ray crystallography.

Ionic liquid biodegradability depends on specific wastewater microbial consortia.

Complete biodegradation of a newly-synthesized chemical in a wastewater treatment plant (WWTP) eliminates the potential for novel environmental pollutants. However, differences within- and between-WWTP microbial communities may alter expectations for biodegradation. WWTP communities can also serve as a source of unique consortia that, when enriched, can metabolize chemicals that tend to resist degradation, but are otherwise promising green alternatives. We tested the biodegradability of three ionic liquids (ILs): 1-octyl-3-methylpyridinium bromide (OMP), 1-butyl-3-methylpyridinium bromide (BMP) and 1-butyl-3-methylimidazolium chloride (BMIM). We performed tests using communities from two WWTPs at three time points. Site-specific and temporal variation both influenced community composition, which impacted the success of OMP biodegradability. Neither BMP nor BMIM degraded in any test, suggesting that these ILs are unlikely to be removed by traditional treatment. Following standard biodegradation assays, we enriched for three consortia that were capable of quickly degrading OMP, BMP and BMIM. Our results indicate WWTPs are not functionally redundant with regard to biodegradation of specific ionic liquids. However, consortia can be enriched to degrade chemicals that fail biodegradability assays. This information can be used to prepare pre-treatment procedures and prevent environmental release of novel pollutants.

Enzymatic de novo pyrimidine nucleotide synthesis.

The use of stable isotope labeling has revolutionized NMR studies of nucleic acids, and there is a need for methods of incorporation of specific isotope labels to facilitate specific NMR experiments and applications. Enzymatic synthesis offers an efficient and flexible means to synthesize nucleoside triphosphates from a variety of commercially available specifically labeled precursors, permitting isotope labeling of RNAs prepared by in vitro transcription. Here, we recapitulate de novo pyrimidine biosynthesis in vitro, using recombinantly expressed enzymes to perform efficient single-pot syntheses of UTP and CTP that bear a variety of stable isotope labeling patterns. Filtered NMR experiments on (13)C, (15)N, (2)H-labeled HIV-2 TAR RNA demonstrate the utility and value of this approach. This flexible enzymatic synthesis will make implementing detailed and informative RNA stable isotope labeling schemes substantially more cost-effective and efficient, providing advanced tools for the study of structure and dynamics of RNA molecules.

Nitric-oxide synthase forms N-NO-pterin and S-NO-cys: implications for activity, allostery, and regulation.

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS(4) site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.

Structure of the GLD-1 homodimerization domain: insights into STAR protein-mediated translational regulation.

Posttranscriptional regulation of gene expression is an important mechanism for modulating protein levels in eukaryotes, especially in developmental pathways. The highly conserved homodimeric STAR/GSG proteins play a key role in regulating translation by binding bipartite consensus sequences in the untranslated regions of target mRNAs, but the exact mechanism remains unknown. Structures of STAR protein RNA binding subdomains have been determined, but structural information is lacking for the homodimerization subdomain. Here, we present the structure of the C. elegans GLD-1 homodimerization domain dimer, determined by a combination of X-ray crystallography and NMR spectroscopy, revealing a helix-turn-helix monomeric fold with the two protomers stacked perpendicularly. Structure-based mutagenesis demonstrates that the dimer interface is not easily disrupted, but the structural integrity of the monomer is crucial for GLD-1 dimerization. Finally, an improved model for STAR-mediated translational regulation of mRNA, based on the GLD-1 homodimerization domain structure, is presented.

Enzymatic synthesis and structural characterization of 13C, 15N-poly(ADP-ribose).

Poly(ADP-ribose) is a significant nucleic acid polymer involved with diverse functions in eukaryotic cells, yet no structural information is available. A method for the synthesis of (13)C, (15)N-poly(ADP-ribose) (PAR) has been developed to allow characterization of the polymer using multidimensional nuclear magnetic resonance (NMR) spectroscopy. Successful integration of pentose phosphate, nicotinamide adenine dinucleotide biosynthesis, and cofactor recycling pathways with poly(ADP-ribose) polymerase-1 permitted labeling of PAR from (13)C-glucose and (13)C, (15)N-ATP in a single pot reaction. The scheme is efficient, yielding approximately 400 nmoles of purified PAR from 5 mumoles ATP, and the behavior of the synthetic PAR is similar to data from PAR synthesized by cell extracts. The resonances for (13)C, (15)N-PAR were unambiguously assigned, but the polymer appears to be devoid of inherent regular structure. PAR may form an ordered macromolecular structure when interacting with proteins, and due to the extensive involvement of PAR in cell function and disease, further studies of PAR structure will be required. The labeled PAR synthesis reported here will provide an essential tool for the future study of PAR-protein complexes.

Synergy of NMR, computation, and X-ray crystallography for structural biology.

NMR spectroscopy and X-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structures at high resolution. More recently, significant advances have been made in algorithms for the de novo prediction of protein structure, and, in favorable cases, the predicted models agree extremely well with experimentally determined structures. Here, we demonstrate a synergistic combination of NMR spectroscopy, de novo structure prediction, and X-ray crystallography in an effective overall strategy for rapidly determining the structure of the coat protein C-terminal domain from the Sulfolobus islandicus rod-shaped virus (SIRV). This approach takes advantage of the most accessible aspects of each structural technique and may be widely applicable for structure determination.

Pathway engineered enzymatic de novo purine nucleotide synthesis.

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.

Crystal structure of Lsm3 octamer from Saccharomyces cerevisiae: implications for Lsm ring organisation and recruitment.

Sm and Sm-like (Lsm) proteins are core components of the ribonucleoprotein complexes essential to key nucleic acid processing events within the eukaryotic cell. They assemble as polyprotein ring scaffolds that have the capacity to bind RNA substrates and other necessary protein factors. The crystal structure of yeast Lsm3 reveals a new organisation of the L/Sm beta-propeller ring, containing eight protein subunits. Little distortion of the characteristic L/Sm fold is required to form the octamer, indicating that the eukaryotic Lsm ring may be more pliable than previously thought. The homomeric Lsm3 octamer is found to successfully recruit Lsm6, Lsm2 and Lsm5 directly from yeast lysate. Our crystal structure shows the C-terminal tail of each Lsm3 subunit to be engaged in connections across rings through specific beta-sheet interactions with elongated loops protruding from neighbouring octamers. While these loops are of distinct length for each Lsm protein and generally comprise low-complexity polar sequences, several Lsm C-termini comprise hydrophobic sequences suitable for beta-sheet interactions. The Lsm3 structure thus provides evidence for protein-protein interactions likely utilised by the highly variable Lsm loops and termini in the recruitment of RNA processing factors to mixed Lsm ring scaffolds. Our coordinates also provide updated homology models for the active Lsm[1-7] and Lsm[2-8] heptameric rings.

Solution NMR studies of the maturation intermediates of a 13 MDa viral capsid.

Dynamic regions of proteins often play essential roles in function, assembly, or maturation of macromolecular complexes. When X-ray crystallography and cryo-electron microscopy are used to investigate macromolecular structures, information about these dynamic regions is lost because of conformational disorder or flexibility. Structural studies on the precursor capsid conformations of the lambdoid bacteriophage HK97, a model system for macromolecular maturation, reveal that essential regions tend to be disordered at early maturation stages. In the Prohead II intermediate, the regions that have the greatest disorder are the N-terminal residues and the E-loop, a region involved in the formation of inter-subunit cross-links. The N-terminus of the subunits in the 13 MDa procapsid is sufficiently dynamic to be studied by solution nuclear magnetic resonance (NMR) spectroscopy. NMR measurements enabled the identification and assignment of resonances to specific residues, assessment of the region's behavior within the context of the capsid, and monitoring of changes in these residues during the maturation process. In the precursor Prohead II and immature EI-III states, the N-termini are found to make transient interactions with the interior capsid surface, while at least a subset of N-termini in EI-III becomes more flexible with time. No resonances are observed for the fully mature Head II capsid, which is consistent with its completely ordered structure. NMR spectroscopy complements the current X-ray crystallography and cryo-electron microscopy data of HK97 by providing key information about the behavior of essential dynamic regions only inferred by other techniques.

Structure and function of the PWI motif: a novel nucleic acid-binding domain that facilitates pre-mRNA processing.

The PWI motif is a highly conserved domain of unknown function in the SRm160 splicing and 3'-end cleavage-stimulatory factor, as well as in several other known or putative pre-mRNA processing components. We show here that the PWI motif is a new type of RNA/DNA-binding domain that has an equal preference for single- and double-stranded nucleic acids. Deletion of the motif prevents SRm160 from binding RNA and stimulating 3'-end cleavage, and its substitution with a heterologous RNA-binding domain restores these functions. The NMR solution structure of the SRm160-PWI motif reveals a novel, four-helix bundle and represents the first example of an alpha-helical fold that can bind single-stranded (ss)RNA. Structure-guided mutagenesis indicates that the same surface is involved in RNA and DNA binding and requires the cooperative action of a highly conserved, adjacent basic region. Thus, the PWI motif is a novel type of nucleic acid-binding domain that likely has multiple important functions in pre-mRNA processing, including SRm160-dependent stimulation of 3'-end formation.