Kenneth Satyshur

University of Wisconsin–Madison | UW · Department of Bacteriology, Biomolecular Chemistry, Neuroscience, Oncology, Carbone Cancer Center, and School of Pharmacy

Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (P...

Both bioactive sphingolipids and Sigma-1 receptor (S1R) chaperones occur ubiquitously in mammalian cell membranes. Endogenous compounds that regulate the S1R are important for controlling S1R responses to cellular stress. Herein, we interrogated the S1R in intact Retinal Pigment Epithelial cells (ARPE-19) with the bioactive sphingoid base, sphingos...

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zin...

Transcription regulation is a key process in all living organisms, involving a myriad of transcription factors. In E. coli , the regulator of the iron-sulfur cluster biogenesis pathway, IscR, acts as a global transcription factor, activating the transcription of some pathways and repressing others.

First synthesized in the 1950s, benzodiazepines are widely prescribed drugs that exert their anxiolytic, sedative and anticonvulsant actions by binding to GABA-A receptors, the main inhibitory ligand-gated ion channel in the brain. Scientists have long theorized that there exists an endogenous benzodiazepine, or endozepine, in the brain. While ther...

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we...

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a thi...

The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates wit...

Phenylalanine and cysteine comprise common miss-sense variants (i.e., single nucleotide polymorphisms [SNPs]) at amino acid position 254 of the human indole(ethyl)amine-N-methyltransferase (hINMT). The phenylalanine variant, which occurs in linkage disequilibrium with two 3’ UTR SNPs, has been reported to associate with elevated urine levels of tri...

Phenol-soluble modulin α3 (PSMα3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSMα3-mediated toxicity. One hypothesis is that PSMα3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSMα3, possibly oligomer...

The signaling ligand (p)ppGpp binds diverse targets across bacteria, yet the mechanistic and evolutionary basis underlying these ligand-protein interactions remains poorly understood. Here we identify a novel (p)ppGpp binding motif in the enzyme HPRT, where (p)ppGpp shares identical binding residues for PRPP and nucleobase substrates to regulate pu...

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic like ser/thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically usef...

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA...

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PAS family transcription facto...

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling...

Antibiotic resistant bacteria are an increasing global problem, and pathogenic actinomycetes and firmicutes are particularly challenging obstacles. These pathogens share several eukaryotic-like kinases that present antibiotic development opportunities. We used computational modelling to identify human kinase inhibitors that could be repurposed towa...

Cacodylate and Zinc chelate the His tag on a bacterial response regulator protein.

Aim: The treatment of psoriasis remains elusive, underscoring the need for identifying novel disease targets and mechanism-based therapeutic approaches. We recently reported that the PI3K/Akt/mTOR pathway that is frequently deregulated in many malignancies is also clinically relevant for psoriasis. We also provided rationale for developing delphin...

Importance: BphP histidine kinases and their cognate response regulators comprise widespread red light sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing, and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or resultant phenotypes. To begin...

Genetically encoded fluorescent markers have revolutionized cell and molecular biology due to their biological compatibility, controllable spatiotemporal expression, and photostability. To achieve in vivo imaging in whole animals, longer excitation wavelength probes are needed due to the superior ability of near infrared light to penetrate tissues...

Quasiracemic crystallography has been used to explore the significance of homochiral and heterochiral associations in a set of host-defense peptide derivatives. The previously reported racemic crystal structure of a magainin 2 derivative displayed a homochiral antiparallel dimer association featuring a "phenylalanine zipper" notable for the dual ro...

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combi...

Metadherin (MTDH) and Staphylococcal nuclease domain containing 1 (SND1) are overexpressed and interact in diverse cancer types. The structural mechanism of their interaction remains unclear. Here, we determined the high-resolution crystal structure of MTDH-SND1 complex, which reveals an 11-residue MTDH peptide motif occupying an extended protein g...

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl...

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitt...

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site...

Refolding and analysis of TR4 expressed and purified from E.coli. TR4 migrated at varied Mr under non-reducing conditions (Non-reduced), and chiefly at 10 kD under reducing conditions (Reduced TR4). Refolding conditions and glutathione gradient parameters were adjusted until TR4 eluted from the NiNTA column as a single predominant band (Refolded)....

BAD-1 binding to heparin agarose BAD-1 Heparin binding was quantified by comparing the initial A280 of the purified, soluble BAD-1 to the A280 of BAD-1 in the unbound aqueous phase. (A) Binding of BAD-1 to heparin and alternative resins. Heparin agarose resin pulled down the majority of BAD-1 in this assay (right column). The relative binding of BA...

Supplementary Materials. (DOCX)

Effect of salt and pH on binding of BAD-1 to heparin. Effect of NaCl (A) and pH (B) on binding of BAD-1 to immobilized heparin. A known concentration of BAD-1 was incubated with heparin agarose. The percentage of BAD-1 bound was quantified by measuring A280 of supernate after incubation, compared to that of the starting material. Results are the me...

Recombinant B. dermatitidis yeast displaying BAD-1 derivatives. Yeast were probed with primary anti-BAD-1 monocolonal antibody DD5-CB4 and a secondary GAM-FITC antibody (Sigma) to quantify surface BAD-1 using a FACscan flow cytometer (Becton Dickenson). B. dermatitidis yeast transformed to produce truncated forms of BAD-1 (Trepeat Y and –AE, bearin...

SPR of BAD-1 lacking 20 copies of the tandem repeat (TrepeatΔ20). Interaction of TrepeatΔ20 with heparin measured by surface plasmon resonance (SPR). TrepeatΔ20 binding was monitored using a Biorad Proteon XPR36. TrepeatΔ20 at the indicated concentrations was injected onto Biorad NLC neutravidin surface with biotinylated heparin immobilized to leve...

3-D representation of a model of the tandem repeat heparin-binding domain. (A) The “distal conformation” has the tryptophans of the WxxWxxW motif intercalating with the basic residues of the BxBxB motif distal to the disulfide bond, in contrast to the proximal model. (B) In the “hairpin conformation”, the tryptophans and basic residues intercalate...

Author Summary Work on fungi is of worldwide importance due to the increasing burden of diseases caused by these agents in humans, plants and animals globally and throughout our ecosystem. The human pathogen Blastomyces dermatitidis harbors an essential virulence factor BAD-1. We describe new structural and functional features of BAD-1 that account...

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report c...

Related to Figure 1. Overlay of gel filtration chromatograms of PR70 (108-519) (dashed blue curve), B′ε (dashed orange curve), and the holoenzymes containing PR70 (108-519) (blue curve) and B′ε (orange curve), respectively.

Related to Figure 6. (A) Pull-down of wild type and mutant PR70 (108-575) proteins by GST-Cdc6 (18-90).

Related to Figure 5. Normalized input of PP2A AC core enzyme, AC-PR70 (108-575) holoenzyme, and AC-PR70 (108-519) holoenzyme for phosphatase assay in Figure 5B&C was examined by SDS-PAGE and visualized by Coomassie blue staining.

Related to Figure 7. Electrostatic potential of the PP2A core enzyme (AC heterodimer) and the B′ε and PR70 regulatory subunits and holoenzymes.

Cyclic constraints have proven to be very effective for preorganizing beta-amino acid residues and thereby stabilizing beta- and alpha/beta-peptide helices, but little is known about possible preorganization effects among gamma residues. Here we assess and compare the impact of cyclic preorganization of beta and gamma residues in the context of a s...

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabil...

The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding n...

Supplementary Figures S1-S5 and Supplementary Table S1

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase in all eukaryotic cells, which functions as holoenzyme consisting of catalytic (PP2Ac), regulatory and scaffolding subunits. Formation of PP2A holoenzymes is regulated by post‐translational modifications of the C‐terminus of PP2Ac (C‐tail), most notably carboxyl methylation on Leu‐309 cat...

Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular function via diverse families of heterotrimeric holoenzymes. A regulatory subunit from the B” family plays a role in regulating the cellular level of Cdc6, a cell cycle regulator crucial for precise control of DNA replication licensing. No previous structural information for...

Protein Phosphatase 2A (PP2A) is one of the most important phosphatases essential for many cellular processes. PP2A Phosphatase Activator (PTPA) plays an important role in PP2A activation. The underlying mechanism remained unclear. To determine the structural basis of PP2A activation, we determined the crystal structure of the PP2A‐PTPA‐ATPγS compl...

Phosphoprotein α4 is essential for cell survival and is known as an inhibitor of protein phosphatase 2A (PP2A). α4 binds to PP2A catalytic subunit (PP2Ac) and stabilizes it in an inactive, latent form. Strikingly, we show that α4 binds preferentially to the partially folded PP2Ac. The crystal structure of α4 bound to a folding sub‐domain of PP2Ac (...

The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded...

Purification of RpoB partners. SDS-PAGE analysis of protein complexes purified from GY12809 strain. The tagged protein is RpoB and purification was carried out from exponential cells. * This band corresponds to the tagged RpoB fused with the calmodulin binding protein. (PDF)

MS/MS spectrum of the peptide [METALLTLDTLAK] corresponding to the N-terminus of DR1245 protein. The mass of the parent di-charged ion was measured at m/z 718.3895 (mass error 0.38 ppm) with an LTQ-Orbitrap XL mass spectrometer (Thermo). The annotated secondary b and y ions are indicated. (PDF)

Genome context of deinococcal dr1245 homologous genes. dr1245 homologues from sequenced Deinococcacae (D. radiodurans, dr1245; D. gobiensis, dgo_CA1600, D. deserti, deide_11730; D. geothermalis, dgeo_1085; D. proteolyticus, deipr_0969) were colored in dark blue whereas, the neighbouring proline dipeptidase, lysM, and, oligoendopeptidase F genes wer...

List of peptides identified by tandem mass spectrometry and their characteristics. (XLSX)

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution,...

The aryl hydrocarbon receptor (AHR) is a transcription factor that responds to diverse ligands and plays a critical role in toxicology, immune function, and cardiovascular physiology. The structural basis of the AHR for ligand promiscuity and preferences is critical for understanding AHR function. Based on the structure of a closely related protein...

Quasiracemic crystallization has been used to obtain high-resolution structures of two variants of the villin headpiece subdomain (VHP) that contain a pentafluorophenylalanine (F(5)Phe) residue in the hydrophobic core. In each case, the crystal contained the variant constructed from l-amino acids and the native sequence constructed from d-amino aci...

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, the flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comp...

Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically avai...

TGF-β-induced expression levels of five endogenous genes in C2C12 cells are increased by exogenous wild-type Smad3 but different mutant Smad3s exhibit a variety of alterations in the basal and TGF-β-induced expression levels. RNA was isolated from cell lysates prepared from two independent sets (A and B) of C2C12 cell populations independently infe...

Mutations on Smad3 MH2 have differential effects on interactions with Smad-binding proteins. Binding between wild-type or mutant Renilla-Smad3 fusion proteins and nine different Flag epitope-tagged Smad-binding proteins was quantified by pull-down of protein complexes from cell lysates and detection of the Renilla luciferase activity per well. The...

Effects of single alanine mutations in the Smad3 MH2 region implicated in binding to Ski on interactions with Smad3-binding proteins. Binding between wild-type or mutant Renilla-Smad3 fusion proteins and eight different Flag epitope-tagged Smad-binding proteins was quantified by pull-down of protein complexes from cell lysates and detection of the...

Primer and probe sequences used for RT and Q-RT-PCR. Primer and probe (where applicable) sequences used for RT and Q-RT-PCR. Probes were labeled with 5′ FAM and 3′ Iowa Black with an internal ZEN quencher. (XLS)

Background: Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent...

Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crysta...

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Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize bioch...