Discovery of an NAD+ analogue with enhanced specificity for PARP1.

Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD+). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD+ featuring an adenosyl 3'-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoform-specific modulators.

Cryo-EM structure of human GPR158 receptor coupled to the RGS7-Gß5 signaling complex.

GPR158 is an orphan G protein­coupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryo­electron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.

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

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.

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

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.

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

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.

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

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.

Unique Polypharmacology Nuclear Receptor Modulator Blocks Inflammatory Signaling Pathways.

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.

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

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/ß5 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.

Activation of Two Sequential H-transfers in the Thymidylate Synthase Catalyzed Reaction.

Thymidylate synthase (TSase) catalyzes the de novo biosynthesis of thymidylate, a precursor for DNA, and is thus an important target for chemotherapeutics and antibiotics. Two sequential C-H bond cleavages catalyzed by TSase are of particular interest: a reversible proton abstraction from the 2'-deoxy-uridylate substrate, followed by an irreversible hydride transfer forming the thymidylate product. QM/MM calculations of the former predicted a mechanism where the abstraction of the proton leads to formation of a novel nucleotide-folate intermediate that is not covalently bound to the enzyme (Wang, Z.; Ferrer, S.; Moliner, V.; Kohen, A. Biochemistry2013, 52, 2348-2358). Existence of such intermediate would hold promise as a target for a new class of drugs. Calculations of the subsequent hydride transfer predicted a concerted H-transfer and elimination of the enzymatic cysteine (Kanaan, N.; Ferrer, S.; Marti, S.; Garcia-Viloca, M.; Kohen, A.; Moliner, V. J. Am. Chem. Soc.2011, 133, 6692-6702). A key to both C-H activations is a highly conserved arginine (R166) that stabilizes the transition state of both H-transfers. Here we test these predictions by studying the R166 to lysine mutant of E. coli TSase (R166K) using intrinsic kinetic isotope effects (KIEs) and their temperature dependence to assess effects of the mutation on both chemical steps. The findings confirmed the predictions made by the QM/MM calculations, implicate R166 as an integral component of both reaction coordinates, and thus provide critical support to the nucleotide-folate intermediate as a new target for rational drug design.

Concerted versus stepwise mechanism in thymidylate synthase.

Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.