Publications by authors named "Scott J Novick"

30 Publications

  • Page 1 of 1

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

J Mol Biol 2021 Sep 18:167258. Epub 2021 Sep 18.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida, USA. Electronic address:

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to 'sense' the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.
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http://dx.doi.org/10.1016/j.jmb.2021.167258DOI Listing
September 2021

One-step construction of circularized nanodiscs using SpyCatcher-SpyTag.

Nat Commun 2021 Sep 14;12(1):5451. Epub 2021 Sep 14.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida, USA.

Circularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. In particular, cNDs can stabilize large patches of lipid bilayers for the reconstitution of complex membrane biochemical reactions, enabling the capture of crucial intermediates involved in synaptic transmission and viral entry. However, previous methods for building cNDs require multiple steps and suffer from low yields. We herein introduce a simple, one-step approach to ease the construction of cNDs using the SpyCatcher-SpyTag technology. This approach increases the yield of cNDs by over 10-fold and is able to rapidly generates cNDs with diameters ranging from 11 to over 100 nm. We demonstrate the utility of these cNDs for mechanistic interrogations of vesicle fusion and protein-lipid interactions that are unattainable using small nanodiscs. Together, the remarkable performance of SpyCatcher-SpyTag in nanodisc circularization paves the way for the use of cNDs in membrane biochemistry and structural biology.
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http://dx.doi.org/10.1038/s41467-021-25737-7DOI Listing
September 2021

Structure of an AMPK complex in an inactive, ATP-bound state.

Science 2021 07;373(6553):413-419

Department of Structural Biology, Van Andel Institute, Grand Rapids, MI 49503, USA.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
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http://dx.doi.org/10.1126/science.abe7565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428800PMC
July 2021

Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay.

Bioorg Med Chem 2021 Jul 6;42:116246. Epub 2021 Jun 6.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, United States. Electronic address:

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
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http://dx.doi.org/10.1016/j.bmc.2021.116246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279689PMC
July 2021

Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.

Nat Commun 2021 05 28;12(1):3239. Epub 2021 May 28.

Department of Integrative Structural and Computational Biology, Scripps Research, La Jolla, CA, USA.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
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http://dx.doi.org/10.1038/s41467-021-23495-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163871PMC
May 2021

Structure-Activity Relationship and Biological Investigation of SR18292 (), a Suppressor of Glucagon-Induced Glucose Production.

J Med Chem 2021 01 12;64(2):980-990. Epub 2021 Jan 12.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida 33458, United States.

Despite a myriad of available pharmacotherapies for the treatment of type 2 diabetes (T2D), challenges still exist in achieving glycemic control. Several novel glucose-lowering strategies are currently under clinical investigation, highlighting the need for more robust treatments. Previously, we have shown that suppressing peroxisome proliferator-activated receptor gamma coactivator 1-alpha activity with a small molecule (SR18292, ) can reduce glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Despite structural similarities in to known β-blockers, detailed structure-activity relationship studies described herein have led to the identification of analogues lacking β-adrenergic activity that still maintain the ability to suppress glucagon-induced glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Hence, these compounds exert their biological effects in a mechanism that does not include adrenergic signaling. These probe molecules may lead to a new therapeutic approach to treat T2D either as a single agent or in combination therapy.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869975PMC
January 2021

Comparative Analysis of Cleavage Specificities of Immobilized Porcine Pepsin and Nepenthesin II under Hydrogen/Deuterium Exchange Conditions.

Anal Chem 2020 08 30;92(16):11018-11028. Epub 2020 Jul 30.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida 33458, United States.

Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.
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http://dx.doi.org/10.1021/acs.analchem.9b05694DOI Listing
August 2020

Definition of functionally and structurally distinct repressive states in the nuclear receptor PPARγ.

Nat Commun 2019 12 20;10(1):5825. Epub 2019 Dec 20.

Department of Biomedical and Pharmaceutical Sciences, University of Montana, 32 Campus Drive, Missoula, MT, 59812, USA.

The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.
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http://dx.doi.org/10.1038/s41467-019-13768-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925260PMC
December 2019

HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists.

Elife 2019 06 7;8. Epub 2019 Jun 7.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States.

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it's unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.
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http://dx.doi.org/10.7554/eLife.47172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579513PMC
June 2019

Unique Polypharmacology Nuclear Receptor Modulator Blocks Inflammatory Signaling Pathways.

ACS Chem Biol 2019 05 15;14(5):1051-1062. Epub 2019 Apr 15.

Department of Molecular Medicine , The Scripps Research Institute , Jupiter , Florida 33458 , United States.

Obesity and rheumatic disease are mechanistically linked via chronic inflammation. The orphan receptor TREM-1 (triggering receptor expressed on myeloid cells-1) is a potent amplifier of proinflammatory and noninfectious immune responses. Here, we show that the pan modulator SR1903 effectively blocks TREM-1 activation. SR1903 emerged from a chemical series of potent RORγ inverse agonists, although unlike close structural analogues, it has modest agonist activity on LXR and weak repressive activity (inverse agonism) of PPARγ, three receptors that play essential roles in inflammation and metabolism. The anti-inflammatory and antidiabetic efficacy of this unique modulator in collagen-induced arthritis and diet-induced obesity mouse models is demonstrated. Interestingly, in the context of obesity, SR1903 aided in the maintenance of the thymic homeostasis unlike selective RORγ inverse agonists. SR1903 was well-tolerated following chronic administration, and combined, these data suggest that it may represent a viable strategy for treatment of both metabolic and inflammatory disease. More importantly, the ability of SR1903 to block LPS signaling suggests the potential utility of this unique polypharmacological modulator for treatment of innate immune response disorders.
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http://dx.doi.org/10.1021/acschembio.9b00236DOI Listing
May 2019

HDX-MS reveals dysregulated checkpoints that compromise discrimination against self RNA during RIG-I mediated autoimmunity.

Nat Commun 2018 12 18;9(1):5366. Epub 2018 Dec 18.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA.

Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
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http://dx.doi.org/10.1038/s41467-018-07780-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299088PMC
December 2018

Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors.

Cell 2018 12;175(7):1756-1768.e17

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA. Electronic address:

Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.
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http://dx.doi.org/10.1016/j.cell.2018.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298040PMC
December 2018

Structural organization of a major neuronal G protein regulator, the RGS7-Gβ5-R7BP complex.

Elife 2018 12 12;7. Epub 2018 Dec 12.

Department of Neuroscience, The Scripps Research Institute, Jupiter, United States.

Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.
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http://dx.doi.org/10.7554/eLife.42150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310461PMC
December 2018

Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states.

J Biol Chem 2019 01 26;294(3):953-967. Epub 2018 Nov 26.

From the Center of Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503,

AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the β-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including β-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant αβγ isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK αβγ bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.
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http://dx.doi.org/10.1074/jbc.RA118.004883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341387PMC
January 2019

Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.

Cell 2018 09;175(1):212-223.e17

Laboratory of Genetics, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA; Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
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http://dx.doi.org/10.1016/j.cell.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179368PMC
September 2018

Discovery of Hydrolysis-Resistant Isoindoline N-Acyl Amino Acid Analogues that Stimulate Mitochondrial Respiration.

J Med Chem 2018 04 22;61(7):3224-3230. Epub 2018 Mar 22.

Department of Molecular Medicine , The Scripps Research Institute , Jupiter , Florida 33458 , United States.

N-Acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. We found that administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure, indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis and mitochondrial uncoupling bioactivity of lipidated N-acyl amino acids and their unnatural analogues. Unsaturated fatty acid chains of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of unnatural N-acyl amino acid analogues, characterized by isoindoline-1-carboxylate head groups (37), were resistant to enzymatic degradation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335027PMC
April 2018

Structural and Dynamic Elucidation of a Non-acid PPAR Partial Agonist: SR1988.

Nucl Receptor Res 2018 ;5

Institute for Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.

Targeting peroxisome proliferator-activated receptor (PPAR) by synthetic compounds has been shown to elicit insulin sensitising properties in type 2 diabetics. Treatment with a class of these compounds, the thiazolidinediones (TZDs), has shown adverse side effects such as weight gain, fluid retention, and congestive heart failure. This is due to their full agonist properties on the receptor, where a number of genes are upregulated beyond normal physiological levels. Lessened transactivation of PPAR by partial agonists has proved beneficial in terms of reducing side effects, while still maintaining insulin sensitising properties. However, some partial agonists have been associated with unfavourable pharmacokinetic profiles due to their acidic moieties, often causing partitioning to the liver. Here we present SR1988, a new partial agonist with favourable non-acid chemical properties. We used a combination of X-ray crystallography and hydrogen/deuterium exchange (HDX) to elucidate the structural basis for reduced activation of PPAR by SR1988. This structural analysis reveals a mechanism that decreases stabilisation of the AF2 coactivator binding surface by the ligand.
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http://dx.doi.org/10.11131/2018/101350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428214PMC
January 2018

HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions.

Nat Commun 2017 10 13;8(1):923. Epub 2017 Oct 13.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA.

The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.
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http://dx.doi.org/10.1038/s41467-017-00978-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640644PMC
October 2017

Nucleotide Binding to ARL2 in the TBCD∙ARL2∙β-Tubulin Complex Drives Conformational Changes in β-Tubulin.

J Mol Biol 2017 11 29;429(23):3696-3716. Epub 2017 Sep 29.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, United States. Electronic address:

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.
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http://dx.doi.org/10.1016/j.jmb.2017.09.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693757PMC
November 2017

Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing.

J Biol Chem 2017 07 14;292(30):12653-12666. Epub 2017 Jun 14.

Laboratories of Structural Sciences and Structural Biology and Biochemistry, Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503. Electronic address:

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.
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http://dx.doi.org/10.1074/jbc.M117.793018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535039PMC
July 2017

N-Arylsulfonyl Indolines as Retinoic Acid Receptor-Related Orphan Receptor γ (RORγ) Agonists.

ChemMedChem 2016 Dec 23;11(23):2607-2620. Epub 2016 Nov 23.

Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL, 33458, USA.

The nuclear retinoic acid receptor-related orphan receptor γ (RORγ; NR1F3) is a key regulator of inflammatory gene programs involved in T helper 17 (T 17) cell proliferation. As such, synthetic small-molecule repressors (inverse agonists) targeting RORγ have been extensively studied for their potential as therapeutic agents for various autoimmune diseases. Alternatively, enhancing T 17 cell proliferation through activation (agonism) of RORγ may boost an immune response, thereby offering a potentially new approach in cancer immunotherapy. Herein we describe the development of N-arylsulfonyl indolines as RORγ agonists. Structure-activity studies reveal a critical linker region in these molecules as the major determinant for agonism. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) analysis of RORγ-ligand complexes help rationalize the observed results.
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http://dx.doi.org/10.1002/cmdc.201600491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158182PMC
December 2016

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Anal Chem 2016 06 13;88(12):6607-14. Epub 2016 Jun 13.

The Scripps Research Institute , Department of Molecular Therapeutics, 130 Scripps Way, Jupiter, Florida 33458, United States.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
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http://dx.doi.org/10.1021/acs.analchem.6b01650DOI Listing
June 2016

Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation.

Elife 2016 Feb 2;5. Epub 2016 Feb 2.

Ben May Department for Cancer Research, University of Chicago, Chicago, United States.

Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition.
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http://dx.doi.org/10.7554/eLife.12792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821807PMC
February 2016

Synthetic RORγt Agonists Enhance Protective Immunity.

ACS Chem Biol 2016 Apr 25;11(4):1012-8. Epub 2016 Jan 25.

Department of Molecular Therapeutics, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.

The T cell specific RORγ isoform RORγt has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 and TC17 cells, cells that have demonstrated antitumor efficacy. RORγt controls gene networks that enhance immunity including increased IL17 production and decreased immune suppression. Both synthetic and putative endogenous agonists of RORγt have been shown to increase the basal activity of RORγt enhancing TH17 cell proliferation. Here, we show that activation of RORγt using synthetic agonists drives proliferation of TH17 cells while decreasing levels of the immune checkpoint protein PD-1, a mechanism that should enhance antitumor immunity while blunting tumor associated adaptive immune resistance. Interestingly, putative endogenous agonists drive proliferation of TH17 cells but do not repress PD-1. These findings suggest that synthetic agonists of RORγt should activate TC17/TH17 cells (with concomitant reduction in the Tregs population), repress PD-1, and produce IL17 in situ (a factor associated with good prognosis in cancer). Enhanced immunity and blockage of immune checkpoints has transformed cancer treatment; thus such a molecule would provide a unique approach for the treatment of cancer.
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http://dx.doi.org/10.1021/acschembio.5b00899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5178133PMC
April 2016

Pharmacological repression of PPARγ promotes osteogenesis.

Nat Commun 2015 Jun 12;6:7443. Epub 2015 Jun 12.

Departments of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter FL33458, USA.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently, we reported the development of PPARγ antagonist SR1664, designed to block the obesity-induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin-sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow-derived mesenchymal stem cells with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand-mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.
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http://dx.doi.org/10.1038/ncomms8443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471882PMC
June 2015

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Proc Natl Acad Sci U S A 2014 Nov 3;111(46):16436-41. Epub 2014 Nov 3.

Codexis, Inc., Redwood City, CA 94063;

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.
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http://dx.doi.org/10.1073/pnas.1411461111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246266PMC
November 2014

Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids.

J Am Chem Soc 2006 Aug;128(33):10923-9

BioCatalytics, Inc., 129 North Hill Avenue, Suite 103, Pasadena, California 91106, USA.

Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity toward the d-enantiomer (95 to >99% ee) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis.
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http://dx.doi.org/10.1021/ja0603960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533268PMC
August 2006

Protein-containing hydrophobic coatings and films.

Biomaterials 2002 Jan;23(2):441-8

Department of Chemical and Biochemical Engineering, The University of Iowa, Iowa City 52242, USA.

The incorporation of enzymes and other proteins into hydrophobic polymeric coatings and films has been investigated in this study with the goal of generating biologically active materials for biocatalysis, antifouling surfaces, and biorecognition. The protein-polymer composites are created using standard solution coating techniques with poly(methyl methacrylate), polystyrene, and poly(vinyl acetate) as polymers and alpha-chymotrypsin and trypsin as biocatalysts. The specific enzyme is first extracted into a nonpolar organic solvent using hydrophobic ion-pairing. The ion-paired enzyme is dried and redissolved into a solvent also miscible with the polymer. This solution is then poured over a surface and the solvent is allowed to evaporate to form the enzyme-containing coating, which can then be delaminated to form a film. Leaching of enzyme from and activity of the biocatalytic coatings and films were evaluated. The biocatalytic coatings showed no loss of activity over ca. one week. For the biocatalytic films, the leaching rate was initially high followed by a slow rate of enzyme loss. Activity was measurable for at least one month, with only ca. one-third of the initial activity lost in that time, while, being continuously incubated in a buffer solution. Activity was also exhibited on macromolecular (protein) substrates. The biocatalytic coatings could be reused over 100 times with only a modest loss of activity. Finally, coatings and films containing a lectin (Concanavalin A) were capable of selectively binding to glycoproteins, thereby extending the application of such films for use in bioseparations and biorecognition.
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http://dx.doi.org/10.1016/s0142-9612(01)00123-5DOI Listing
January 2002
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