Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins.

The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

The Sts Proteins: Modulators of Host Immunity.

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.

Human uridine 5'-monophosphate synthase stores metabolic potential in inactive biomolecular condensates.

Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.

Structure of Klebsiella pneumoniae adenosine monophosphate nucleosidase.

Klebsiella pneumoniae is a bacterial pathogen that is increasingly responsible for hospital-acquired pneumonia and sepsis. Progressive development of antibiotic resistance has led to higher mortality rates and creates a need for novel treatments. Because of the essential role that nucleotides play in many bacterial processes, enzymes involved in purine and pyrimidine metabolism and transport are ideal targets for the development of novel antibiotics. Herein we describe the structure of K. pneumoniae adenosine monophosphate nucleosidase (KpAmn), a purine salvage enzyme unique to bacteria, as determined by cryoelectron microscopy. The data detail a well conserved fold with a hexameric overall structure and clear density for the putative active site residues. Comparison to the crystal structures of homologous prokaryotic proteins confirms the presence of many of the conserved structural features of this protein yet reveals differences in distal loops in the absence of crystal contacts. This first cryo-EM structure of an Amn enzyme provides a basis for future structure-guided drug development and extends the accuracy of structural characterization of this family of proteins beyond this clinically relevant organism.

The Intersection of Purine and Mitochondrial Metabolism in Cancer.

Nucleotides are essential to cell growth and survival, providing cells with building blocks for DNA and RNA, energy carriers, and cofactors. Mitochondria have a critical role in the production of intracellular ATP and participate in the generation of intermediates necessary for biosynthesis of macromolecules such as purines and pyrimidines. In this review, we highlight the role of purine and mitochondrial metabolism in cancer and how their intersection influences cancer progression, especially in ovarian cancer. Additionally, we address the importance of metabolic rewiring in cancer and how the evolving landscape of purine synthesis and mitochondria inhibitors can be potentially exploited for cancer treatment.

Structure of the E. coli agmatinase, SPEB.

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 µs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone C═O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Structural Basis for the Regulation of Biofilm Formation and Iron Uptake in A. baumannii by the Blue-Light-Using Photoreceptor, BlsA.

The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a blue-light-using flavin adenine dinucleotide (BLUF) protein that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA-binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photoactivated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin-binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light-dependent modulation of biofilm formation and iron uptake.

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light-Oxygen-Voltage (LOV) Protein.

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.

An unexpected 2-histidine phosphoesterase activity of suppressor of T-cell receptor signaling protein 1 contributes to the suppression of cell signaling.

The suppressor of T-cell receptor (TCR) signaling (Sts) proteins Sts-1 and Sts-2 suppress receptor-mediated signaling pathways in various immune cells, including the TCR pathway in T cells and the Dectin-1 signaling pathway in phagocytes. As multidomain enzymes, they contain an N-terminal ubiquitin-association domain, a central Src homology 3 domain, and a C-terminal histidine phosphatase domain. Recently, a 2-histidine (2H) phosphoesterase motif was identified within the N-terminal portion of Sts. The 2H phosphoesterase motif defines an evolutionarily ancient protein domain present in several enzymes that hydrolyze cyclic phosphate bonds on different substrates, including cyclic nucleotides. It is characterized by two invariant histidine residues that play a critical role in catalytic activity. Consistent with its assignment as a phosphoesterase, we demonstrate here that the Sts-1 2H phosphoesterase domain displays catalytic, saturable phosphodiesterase activity toward the dinucleotide 2',3'-cyclic NADP. The enzyme exhibited a high degree of substrate specificity and selectively generated the 3'-nucleotide as the sole product. Sts-1 also had phosphodiesterase catalytic activity toward a 5-mer RNA oligonucleotide containing a 2',3'-cyclic phosphate group at its 3' terminus. To investigate the functional significance of Sts-1 2H phosphoesterase activity, we generated His-to-Ala variants and examined their ability to negatively regulate cellular signaling pathways. Substitution of either conserved histidine compromised the ability of Sts-1 to suppress signaling pathways downstream of both the TCR and the Dectin-1 receptor. Our results identify a heretofore unknown cellular enzyme activity associated with Sts-1 and indicate that this catalytic activity is linked to specific cell-signaling outcomes.

Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily.

Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.

Vibrational spectroscopy of flavoproteins.

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.

Structure-Based Design, Synthesis, and Biological Evaluation of Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme MenE.

N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 µM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.

Fast, volumetric live-cell imaging using high-resolution light-field microscopy.

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

Crystal structure of E. coli PRPP synthetase.

BACKGROUND: Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) is an enzyme that catalyzes the ATP-dependent conversion of ribose-5-phosphate to phosphoribosyl pyrophosphate. The reaction product is a key precursor for the biosynthesis of purine and pyrimidine nucleotides. RESULTS: We report the 2.2 Å crystal structure of the E. coli ribose-phosphate pyrophosphobinase (EcKPRS). The protein has two type I phosphoribosyltransferase folds, related by 2-fold pseudosymmetry. The propeller-shaped homohexameric structure of KPRS is composed of a trimer of dimers, with the C-terminal domains forming the dimeric blades of the propeller and the N-terminal domains forming the hexameric core. The key, conserved active site residues are well-defined in the structure and positioned appropriately to bind substrates, adenosine monophosphate and ribose-5-phosphate. The allosteric site is also relatively well conserved but, in the EcKPRS structure, several residues from a flexible loop occupy the site where the allosteric modulator, adenosine diphosphate, is predicted to bind. The presence of the loop in the allosteric site may be an additional level of regulation, whereby low affinity molecules are precluded from binding. CONCLUSIONS: Overall, this study details key structural features of an enzyme that catalyzes a critical step in nucleotide metabolism. This work provides a framework for future studies of this important protein and, as nucleotides are critical for viability, may serve as a foundation for the development of novel anti-bacterial drugs.

Discovery and Characterization of Two Classes of Selective Inhibitors of the Suppressor of the TCR Signaling Family of Proteins.

The suppressor of T-cell receptor signaling (Sts) proteins, Sts-1, has recently emerged as a potential immunostimulatory target for drug development. Genetic inactivation of the Sts proteins dramatically increases host survival of systemic infection and leads to improved pathogen clearance. The protein tyrosine phosphatase (PTP) activity of these proteins arises from a C-terminal 2-histidine phosphatase (HP) domain. To identify new inhibitors of the HP activity of Sts-1, we miniaturized a phosphatase assay to a 1536-well format and conducted a 20 580 compound screen. Among the hits were two classes of structurally related compounds, tetracycline variants and sulfonated azo dyes. These hits had low micromolar to nanomolar IC50 values. Orthogonal screening confirmed the validity of these inhibitors and demonstrated that both act competitively on Sts-1 phosphatase activity. When tested on other PTPs, PTP1B and SHP1, it was found that the tetracycline PTP1B, SHP1, the tetracycline variant (doxycycline), and the sulfonated azo dye (Congo red) are selective inhibitors of Sts-1HP, with selectivity indices ranging from 19 to as high as 200. The planar polyaromatic moieties present in both classes of compounds suggested a common binding mode. The mutation of either tryptophan 494 or tyrosine 596, located near the active site of the protein, reduced the Ki of the inhibitors from 3- to 18-fold, indicating that these residues may help to promote the binding of substrates with aromatic groups. This work provides new insights into substrate selectivity mechanisms and describes two classes of compounds that can serve as probes of function or as a basis for future drug discovery.

Correction: Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1006843.].