Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes.

Poly(UG) or 'pUG' dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3' ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Draft genome of the caprolactam-degrading Paenarthrobacter sp. CAP02 isolated from a landfill.

Here, we report the draft genome sequence of Paenarthrobacter sp. CAP02, which metabolizes caprolactam as a sole source of carbon and nitrogen. This bacterium was isolated from Dane County Landfill Site No. 2 in Madison, WI, USA.

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family.

Pyridoxal phosphate (PLP)-dependent enzymes afford access to a variety of non-canonical amino acids (ncAAs), which are premier buildings blocks for the construction of complex bioactive molecules. The vinylglycine ketimine (VGK) subfamily of PLP-dependent enzymes plays a critical role in sulfur metabolism and is home to a growing set of secondary metabolic enzymes that synthesize γ-substituted ncAAs. Identification of VGK enzymes for biocatalysis faces a distinct challenge because the subfamily contains both desirable synthases as well as lyases that break down ncAAs. Some enzymes have both activities, which may contribute to pervasive mis-annotation. To navigate this complex functional landscape, we used a substrate multiplexed screening approach to rapidly measure the substrate promiscuity of 40 homologs in the VGK subfamily. We found that enzymes involved in transsulfuration are less likely to have promiscuous activities and often possess undesirable lyase activity. Enzymes from direct sulfuration and secondary metabolism generally had a high degree of substrate promiscuity. From this cohort, we identified an exemplary γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). This enzyme is thermostable and has high expression (~400 mg protein per L culture), enabling preparative scale synthesis of thioether containing ncAAs. When assayed with l-allylglycine, CahyGS catalyzes a stereoselective γ-addition reaction to afford access to a unique set of γ-methyl branched ncAAs. We determined high-resolution crystal structures of this enzyme that define an open-close transition associated with ligand binding and set the stage for future engineering within this enzyme subfamily.

Harnessing Aromatic-Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification.

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high-resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor-1, which is targeted by osteoporosis drugs. These results should motivate other structure-based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR.

Enzymatic deconstruction of lignocellulosic biomass is crucial to establishment of the renewable biofuel and bioproduct economy. Better understanding of these enzymes, including their catalytic and binding domains, and other features offer potential avenues for improvement. Glycoside hydrolase family 9 (GH9) enzymes are attractive targets because they have members that exhibit exo- and endo-cellulolytic activity, processivity of reaction, and thermostability. This study examines a GH9 from Acetovibrio thermocellus ATCC 27405, AtCelR containing a catalytic domain and a carbohydrate binding module (CBM3c). Crystal structures of the enzyme without substrate, bound to cellohexaose (substrate) or cellobiose (product), show the positioning of ligands to calcium and adjacent residues in the catalytic domain that may contribute to substrate binding and facilitate product release. We also investigated the properties of the enzyme engineered to contain an additional carbohydrate binding module (CBM3a). Relative to the catalytic domain alone, CBM3a gave improved binding for Avicel (a crystalline form of cellulose), and catalytic efficiency (kcat/KM) was improved 40× with both CBM3c and CBM3a present. However, because of the molecular weight added by CBM3a, the specific activity of the engineered enzyme was not increased relative to the native construct consisting of only the catalytic and CBM3c domains. This work provides new insight into a potential role of the conserved calcium in the catalytic domain and identifies contributions and limitations of domain engineering for AtCelR and perhaps other GH9 enzymes.

Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases.

Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.

Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

A broad specificity ß-propeller enzyme from Rhodopseudomonas palustris that hydrolyzes many lactones including γ-valerolactone.

Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca2+-dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this ß-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other ß-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.

An atypical RNA quadruplex marks RNAs as vectors for gene silencing.

The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.

Altered signaling at the PTH receptor via modified agonist contacts with the extracellular domain provides a path to prolonged agonism in vivo.

The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.

Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction.

Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon-carbon bond forming transformations in traditional organic chemistry and nature, relatively few C-C bond forming enzymes have found their way into the biocatalysis toolbox. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstDv2.0, is efficient in a whole-cell biocatalysis format. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstDv2.0 at 2.25 Šreveals the active site and provides a foundation for probing the mechanism of UstD.

Trimer-to-Monomer Disruption Mechanism for a Potent, Protease-Resistant Antagonist of Tumor Necrosis Factor-α Signaling.

Aberrant tumor necrosis factor-α (TNFα) signaling is associated with many inflammatory diseases. The homotrimeric quaternary structure of TNFα is essential for receptor recognition and signal transduction. Previously, we described an engineered α/ß-peptide inhibitor that potently suppresses TNFα activity and resists proteolysis. Here, we present structural evidence that both the α/ß-peptide inhibitor and an all-α analogue bind to a monomeric form of TNFα. Calorimetry data support a 1:1 inhibitor/TNFα stoichiometry in solution. In contrast, previous cocrystal structures involving peptide or small-molecule inhibitors have shown the antagonists engaging a TNFα dimer. The structural data reveal why our inhibitors favor monomeric TNFα. Previous efforts to block TNFα-induced cell death with peptide inhibitors revealed that surfactant additives to the assay conditions cause a more rapid manifestation of inhibitory activity than is observed in the absence of additives. We attributed this effect to a loose surfactant TNFα association that lowers the barrier to trimer dissociation. Here, we used the new structural data to design peptide inhibitors bearing a surfactant-inspired appendage intended to facilitate TNFα trimer dissociation. The appendage modified the time course of protection from cell death.

Investigation of ß-Substitution Activity of O-Acetylserine Sulfhydrolase from Citrullus vulgaris.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes have garnered interest for their ability to synthesize non-standard amino acids (nsAAs). One such class of enzymes, O-acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l-cysteine. Here, we examine the ß-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non-native reactivity with a variety of O-nucleophiles. Optimization of reaction conditions for carboxylate and phenolate substrates led to distinct conditions that were leveraged for the preparative-scale synthesis of nsAAs. We further show this enzyme is capable of C-C bond formation through a ß-alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.

Identification and characterization of a set of monocot BAHD monolignol transferases.

Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as important "monomers" for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants.

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme.

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Šresolution, which shows an Fe and three-histidine (3-His) active site situated at the end of a wide substrate access channel. The open approach to the active site is consistent with the recent discovery that ADO catalyzes not only the conversion of cysteamine to hypotaurine but also the oxidation of N-terminal cysteine (Nt-Cys) peptides to their corresponding sulfinic acids as part of the eukaryotic N-degron pathway. Whole-protein models of ADO in complex with either cysteamine or an Nt-Cys peptide, generated using molecular dynamics and quantum mechanics/molecular mechanics calculations, suggest occlusion of access to the active site by peptide substrate binding. This finding highlights the importance of a small tunnel that leads from the opposite face of the enzyme into the active site, providing a path through which co-substrate O2 could access the Fe center. Intriguingly, the entrance to this tunnel is guarded by two Cys residues that may form a disulfide bond to regulate O2 delivery in response to changes in the intracellular redox potential. Notably, the Cys and tyrosine residues shown to be capable of forming a cross-link in human ADO reside ∼7 Šfrom the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

Epistasis shapes the fitness landscape of an allosteric specificity switch.

Epistasis is a major determinant in the emergence of novel protein function. In allosteric proteins, direct interactions between inducer-binding mutations propagate through the allosteric network, manifesting as epistasis at the level of biological function. Elucidating this relationship between local interactions and their global effects is essential to understanding evolution of allosteric proteins. We integrate computational design, structural and biophysical analysis to characterize the emergence of novel inducer specificity in an allosteric transcription factor. Adaptive landscapes of different inducers of the designed mutant show that a few strong epistatic interactions constrain the number of viable sequence pathways, revealing ridges in the fitness landscape leading to new specificity. The structure of the designed mutant shows that a striking change in inducer orientation still retains allosteric function. Comparing biophysical and functional properties suggests a nonlinear relationship between inducer binding affinity and allostery. Our results highlight the functional and evolutionary complexity of allosteric proteins.

C. elegans germ granules require both assembly and localized regulators for mRNA repression.

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.