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The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

Peroxisome proliferator-activated receptor (PPAR) ligands have been used in clinical therapy to treat metabolic disease since the 1960s. However, these ligands have side effects that restrict their use, thought to be caused, in part, by their broad specificity. Efforts have been made to synthesize new ligands; however, most have failed to pass clinical trials. Here we examine the available crystal structures of PPAR in complex with ligands to identify common ligand design factors for selectivity towards a PPAR subtype. Methods to improve drug-lead identification and optimization and other factors that may contribute to design of a successful PPAR ligand are discussed.

Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s-1 ), specificity constant (626 ± 110.0 mm-1 ·s-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s-1 ) and lowest specificity constant (40.2 ± 4.4 mm-1 ·s-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1, that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, Tuberous Sclerosis Complex.We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving metabolic reprogramming and an increased rate of glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through a differential interaction with AMPK.Our findings suggest that unique ‘bespoke’ combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

SummaryMonitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a receptor binding domain mutation that leads to weaker ACE2 binding capability based on in silico simulation, which concerns a SARS-CoV-2 sample collected on 27thJanuary 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and requires attention from researchers working on vaccine development around the world.HighlightsBased on the currently available genome sequence data, we provided direct genetic evidence that the SARS-COV-2 genome has a much lower mutation rate and genetic diversity than SARS during the 2002-2003 outbreak.The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development.Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history.We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human.We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a sample collected on 27th January 2020.

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.

The cytochrome P450 enzymes execute a range of selective oxidative biotransformations across many biological systems. The bacterial enzyme CYP199A4 catalyzes the oxidative demethylation of 4-methoxybenzoic acid. The benzoic acid moiety of the molecule binds in the active site of the enzyme such that the functional group at the para-position is held close to the heme iron. Therefore, CYP199A4 has the potential to catalyze alternative monooxygenase reactions with different para-substituted benzoic acid substrates such as thioethers and alkylamines. The oxidation of 4-methyl- and 4-ethyl-thiobenzoic acids by CYP199A4 resulted in sulfur oxidation. 4-Ethylthiobenzoic acid sulfoxidation and 4-ethylbenzoic acid hydroxylation by CYP199A4 occurred with high enantioselectivity (>74% enantiomeric excess). By way of contrast, CYP199A4 catalyzed exclusive oxidative N-demethylation over N-oxide formation with 4-methyl- and 4-dimethylaminobenzoic acids. Unexpectedly acetamide formation by CYP199A4 competes with dealkyla...

Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium, M. colombiense, M. kansasii and M . marinum . CYP268A2, from M. marinum , which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight-chain fatty acids (C10-C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and tran s-pseudoionone at a terminal methyl group to yield ( 2E,6E )-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and ( 3E,5E,9E )-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 A with trans -pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis , which shares 41 % sequence identity. The active site is predominantly hydrophobic but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.

Cytosolic thiolase (CT) catalyzes the reversible Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. The reaction cycle proceeds via a ping-pong mechanism involving an acetylated enzyme intermediate and two separate oxyanion holes which stabilize negatively charged reaction intermediates. This is the initial step in the synthesis of ergosterol in the prominent fungal pathogen Aspergillus fumigatus and is essential for the growth and survival of the organism. Here, we present crystal structures of A. fumigatus CT in liganded and apo forms and in a complex with different monovalent cations. Careful observation of the electron density at the active sites of two different afCT structures crystallized in the presence of acetyl-CoA shows that our crystals have trapped various stages of the thiolase catalytic cycle, including two tetrahedral reaction intermediates that have previously eluded structural characterization. Unexpectedly, we have also shown that afCT is activated by monova...

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 D3 (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.

A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches; in silico modeling currently provides the only insight into how ligands block Ser273 phosphorylation. The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research.

Introduction Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL associated with high relapse rates and poor survival. Rearrangements of Janus kinase 2 (JAK2r) are present in approximately 5% and 14% of pediatric and young adult Ph-like ALL cases respectively. The resultant JAK2 gene fusions drive leukemogenesis through constitutive activation of the JAK/STAT signaling pathway and are associated with very poor outcomes in patients with Ph-like ALL. All JAK inhibitors in clinical development are type I inhibitors, which bind in the ATP-binding site of JAK2. A phase II clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-cell ALL cases harboring JAK2 alterations. The development of treatment resistance to targeted inhibitors in other diseases is well documented and often results in disease relapse. Elucidating mechanisms of ruxolitinib resistance in JAK2r ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of therapeutic strategies to overcome or avert resistance. Methods JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 μM ruxolitinib. Sanger sequencing of the RT-PCR amplified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V, and analysis by flow cytometry. Alterations in signaling pathways were determined using phosphoflow cytometry and western blot analysis. Computational modelling of acquired JAK2 mutations and subsequent influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Results In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localized to the ATP/ruxolitinib binding site and conferred resistance to multiple type-I JAK inhibitors, including ruxolitinib, BMS-911543, and AZD-1480 (Table 1). JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868, which binds in an allosteric site of JAK2 in addition to the ATP-binding site. Ruxolitinib resistance correlated with sustained downstream STAT5 activation in the presence of 1 μM ruxolitinib compared with non-mutant ATF7IP-JAK2 Ba/F3 cells. Intracellular phosphoflow cytometry of ruxolitinib-resistant ATF7IP-JAK2 Ba/F3 cells confirmed constitutive activation of JAK/STAT signaling in the presence of 50 nM ruxolitinib, in contrast to non-mutant ATF7IP-JAK2 Ba/F3 cells. Computational modelling suggested that JAK2 p.L983F (Fig. 1D) sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond (Fig. 1B). The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilizing the JAK2 activation loop (Fig1C). Conclusions This study demonstrates that the JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure in the setting of JAK2r ALL, highlighting the importance of monitoring the emergence of mutations within this region. In addition to previously described mutations we identified a novel JAK2 p.G993A mutation that conferred resistance to both type-I and type-II JAK inhibitors. The JAK2 p.G993A mutation was postulated to modulate the stability of a conserved domain. Understanding mechanisms of ruxolitinib resistance, as modelled here, has the potential to inform future drug design and the development therapeutic strategies for this high-risk cohort. Disclosures White: Amgen: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding.

The solvatochromic amino-acids 4-DMNA or 4-DAPA, were separately introduced at position 147, 150 or 151 of a short p21 peptide (141–155) known to bind sliding clamp protein PCNA. The ability of these peptides, 1a-3a and 1b-3b, to act as a turn-on fluorescent sensor for PCNA was then investigated. The 4-DMNA-containing peptides (1a-3a) displayed up to a 40-fold difference in fluorescence between a polar (Tris buffer) and a hydrophobic solvent (dioxane with 5 mM 18-crown-6), while the 4-DAPA-containing peptides (1b-3b) displayed a significantly enhanced (300-fold) increase in fluorescence from Tris buffer to dioxane with 18-crown-6. SPR analysis of the peptides against PCNA revealed that the 151-substituted peptides 3a and 3b interacted specifically with PCNA, with KD values of 921 nM and 1.28 μM, respectively. Analysis of the fluorescence of these peptides in the presence of increasing concentrations of PCNA revealed a 10-fold change in fluorescence for 3a at 2.5 equivalents of PCNA, compared to only a 3.5-fold change in fluorescence for 3b. Peptide 3a is an important lead for development of a PCNA-selective turn-on fluorescent sensor for application as a cell proliferation sensor to investigate diseases such as cancer.

Colorimetric methods are convenient for the determination of inorganic phosphate. However, the acidic conditions required can complicate measurement of ATPase through non-enzymatic ATP hydrolysis. Here we present an optimized antimony-phosphomolybdate microassay for the simple and rapid detection of ATPase activity, with micromolar sensitivity. The low acidity of the color reagent results in no interference for samples containing up to 0.5–5 mM ATP, dependent on the sample volume. The assay is compatible with common assay conditions and was similar in accuracy to an established continuous method. The simplicity of this method makes it ideal for medium to high throughput applications.

Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include rosiglitazone, pioglitazone and rivoglitazone, with the latter being the most potent. The TZDs are only marginally selective for the therapeutic target PPARγ as they also activate PPARα and PPARδ homologues to varying degrees, causing off-target effects. While crystal structures for TZD compounds in complex with PPARγ are available, minimal structural information is available for TZDs bound to PPARα and PPARδ. This paucity of structural information has hampered the determination of precise structural mechanisms involved in TZD selectivity between PPARs. To help address these questions molecular dynamic simulations were performed of rosiglitazone, pioglitazone and rivoglitazone in complex with PPARα, PPARδ, and PPARγ in order to better understand the mechanisms of PPAR selectivity. The simulations revealed that TZD interactions with residues Tyr314 and Phe318 of PPARα and residues Phe291 and Thr253 of PPARδ as well as the omega loop, are key determinants of TZD receptor selectivity. Notably, in this study, we solve the first X-ray crystal structure of rivoglitazone bound to any PPAR. Rivoglitazone forms a unique hydrogen bond network with the residues of the PPARγ co-activator binding surface (known as AF2) and makes more extensive contacts with helix 3 and the β-sheet as compared to model TZD compounds such as rosiglitazone.

Obtaining high-throughput structural characterization of small molecule drug candidates bound to their protein target has been hampered by the challenges of cocrystallizing protein-ligand complexes. These challenges include poor ligand solubility, excess ligand molecules disrupting the homogeneity of the sample to be crystallized, and inefficiency in preparing individual complexes and crystal screens for each drug candidate. Crystallizing apo protein followed by soaking with a solution containing the ligand of interest is a powerful tool for rapidly obtaining structural information of ligands. Here, we describe the process of purifying, crystallizing, and soaking PPARγ ligand binding domain as well as strategies for cocrystallizing ligands that are not amenable to soaking.

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.

Obtaining high-throughput structural characterization of small molecule drug candidates bound to their protein target has been hampered by the challenges of cocrystallizing protein-ligand complexes. These challenges include poor ligand solubility, excess ligand molecules disrupting the homogeneity of the sample to be crystallized, and inefficiency in preparing individual complexes and crystal screens for each drug candidate. Crystallizing apo protein followed by soaking with a solution containing the ligand of interest is a powerful tool for rapidly obtaining structural information of ligands. Here, we describe the process of purifying, crystallizing, and soaking PPARγ ligand binding domain as well as strategies for cocrystallizing ligands that are not amenable to soaking.

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K i 0.22 M) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 g/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth Refereed/Peer-reviewed

Background Actinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract C H bonds and have important roles in natural product biosynthesis. Methods Two members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography. Results CYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate β-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 A. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes. Conclusions The structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different. General significance Multiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K(i) = 0.22 μM) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth.

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 μM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

Introduction TYK2-rearrangements have recently been detected in high-risk acute lymphoblastic leukemia (HR-ALL) cases and are associated with poor outcome. The resultant fusion protein is predominantly driven by Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. Thus, JAK/TYK2 inhibitors (JAKi) are among the most promising targeted therapeutics against these fusions. This was confirmed in aMYB-TYK2mouse model where the induced aggressive B-ALL was effectively targeted by the novel dual SYK/JAKi, cerdulatinib (cerd) (Tavakoliet al.2020, EHA abstract EP353). Despite the clinical benefit of JAKi in myeloproliferative neoplasms, resistance occurs resulting in relapse. Hence it is necessary to identify potential JAKi-mediated resistance mechanisms inTYK2-rearranged B-ALL patients. This study modeled cerd resistance mechanisms to recapitulate possible clinical scenarios. Methods Ba/F3 pro-B cells were retrovirally transduced with a plasmid construct containing theMYB-TYK2fusion gene isolated from an ALL patient. A cerd resistant line (cerdres) was generated by exposure of Ba/F3-MYB-TYK2cells to increasing concentrations of cerd (up to 3µM; clinically achievable plasma level is 1-2µM) over a period of 151 d. IC50 was determined via CellTiter-Glo proliferation assay. Downstream signaling was determined by phospho-flow analysis. Sanger sequencing was performed over theMYB-TYK2fusion gene to identify emergence of mutations. Site directed mutagenesis of theMYB-TYK2fusion construct was used to model an identified mutationin vitro. Computational modeling of theTYK2mutation and cerd docking was performed via ICM-Pro (Molsoft L.C.C.). The effect of long-term exposure to cerd on activation of JAK family kinases was investigated via western blot. A cerd resensitized line (cerdresen) was generated by culturing cerdres Ba/F3-MYB-TYK2cells in cerd free media for 5 weeks. Results Long-term exposure of Ba/F3-MYB-TYK2cells to cerd resulted in resistance with an 8.7-fold increase in IC50 compared to vehicle control cells (DMSO exposed) (IC50=6508 vs 739nM,p=0.001; Figure 1A). A novel mutation in the kinase domain ofTYK2(p.R987Q, c.3338G>A) was identified in ~50% of cerdres Ba/F3-MYB-TYK2cells. However,de novointroduction ofMYB-TYK2p.R987Q into parental Ba/F3 cells indicated that resistance to cerd was not due to the mutation alone, as these cells displayed no significant decreased sensitivity to cerd compared with control cells (IC50=1200 vs 739nM,p>0.05; Figure A). Computational modeling indicated the binding orientation of cerd to the mutated kinase domain was reversed 180º resulting in less favourable binding (TYK2p.R987Q vsTYK2, binding score= -18.6 vs -23.4). Phosphoflow analysis demonstrated increased JAK/STAT signalling in cerdres Ba/F3-MYB-TYK2compared with control cells (MFI= 35.2 vs 16.5,p=0.008) that persisted despite TYK2 kinase inhibition (MFI= 29.8 vs 2.3,p=0.004; Figure B). Expression of p.R987Q mutation did not result in increased pSTAT5 levels in Ba/F3-MYB-TYK2p.R987Q. Given that JAK2 heterodimerisation with other JAK proteins can lead to JAK/STAT activation and drug persistence (Meyeret al.2017), other kinases may facilitate phosphorylation of TYK2 in cerdres Ba/F3-MYB-TYK2.Western blot analysis confirmed a significant increase in TYK2 phosphorylation (p=0.01) and JAK1 expression (p=0.0008) in cerdres vs control Ba/F3-MYB-TYK2cells (Figure C). Cerd withdrawal resulted in potential resensitization of cerdres Ba/F3-MYB-TYK2to cerd with an associated decrease in IC50 (cerdres vs cerdresen, 6508 vs 2603nM,p=0.0003). However, IC50 levels did not decrease to levels observed in control cells (cerdresen vs control, 2603 vs 739nM, p=0.01), potentially due to increased activation of STAT5 from cerd-induced accumulation of pTYK2 (Tvorogovet al. 2018). Conclusions In vitromodeling suggests that persistent JAK/STAT activation is due to changes in TYK2 expression. Possible heterodimer formation with JAK1 in the setting of JAKi -cerd- exposure allows cells to become resistant. Consequently, the novel evidence of resistance mechanisms to JAKi, provide a rationale for the use of other small molecule inhibitors (e.g. HSP90i and HDACi), to potentially retain TYK2 degradation ability in resistant cells. This targeted approach may contribute to the treatment of patient withTYK2-rearranged ALL. Disclosures White: Bristol-Myers Squibb:Honoraria, Research Funding;Amgen:Honoraria.

The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease., Graphical abstract Image 1, Highlights • PPARγ S273 regulates osteoclast differentiation and insulin sensitivity • PPARγ S112 regulates osteoblast and adipocyte differentiation • PPARγ and PPARα regulate osteocyte activities of bone formation and turnover • SR10171, a PPARγ inverse agonist and PPARα weak agonist, is anabolic for bone Diabetes is a condition with compromised energy balance and is associated with bone fractures. Some treatment options for diabetes sensitize the patient to insulin via targeting the transcription factor PPARγ. PPARγ is also key regulator of bone formation and bone resorption. Anti-diabetic drugs TZDs target PPARγ protein and this leads to bone loss and increase in fractures in postmenopausal women. Bone mass and energy metabolism share similar regulating pathways, and here we demonstrate a new class of insulin sensitizers that is a selective modulator of PPARγ activity; resulting in a pharmacologic agent that can be beneficial for both diabetes and metabolic bone disease.

Here we describe an NMR and X-ray crystallography-based characterisation of the mechanism by which a new class of macrocyclic peptidomimetic aldehyde inhibits α-chymotrypsin. In particular, a (13)C-labelled analogue of the inhibitor was prepared and used in NMR experiments to confirm formation of a hemiacetal intermediate on binding with α-chymotrypsin. Analysis of an X-ray crystallographic structure in complex with α-chymotrypsin reveals that the backbone adopts a stable β-strand conformation as per its design. Binding is further stabilised by interaction with the oxyanion hole near the S1 subsite and multiple hydrogen bonds.

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efficient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with different meta-substituted benzoic acids in order to establish which criteria are important for efficient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efficiently oxidized. For example, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modified position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the C H bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These findings were confirmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efficient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic effect on the activity.

The human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 3

Summary: Peroxisome proliferator activated receptor γ (PPARγ) is a nuclear receptor and target for antidiabetics that increase insulin sensitivity. Owing to the side effects of PPARγ full agonists, research has recently focused on non-activating ligands of PPARγ, which increase insulin sensitivity with decreased side effects. Here, we present the crystal structures of inverse agonist SR10171 and a chemically related antagonist SR11023 bound to the PPARγ ligand-binding domain, revealing an allosteric switch in the activation helix, helix 12 (H12), forming an antagonist conformation in the receptor. H12 interacts with the antagonists to become fixed in an alternative location. Native mass spectrometry indicates that this prevents contacts with coactivator peptides and allows binding of corepressor peptides. Antagonists of related nuclear receptors act to sterically prevent the active configuration of H12, whereas these antagonists of PPARγ alternatively trap H12 in an inactive configuration, which we have termed the tumble and trap mechanism. : Biological Sciences; Endocrinology; Structural Biology Subject Areas: Biological Sciences, Endocrinology, Structural Biology

Targeting peroxisome proliferator-activated receptor γ (PPARγ) 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 PPARγ 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 PPARγ by SR1988. This structural analysis reveals a mechanism that decreases stabilisation of the AF2 coactivator binding surface by the ligand.

The penultimate step in the biosynthesis of biotin is the closure of the ureido heterocycle in a reaction requiring a nucleoside triphosphate (NTP). In Mycobacterium tuberculosis this reaction is catalyzed by dethiobiotin synthetase (MtDTBS). MtDTBS is unusual as it can employ multiple (NTPs), with a >100-fold preference for cytidine triphosphate (CTP). Here the molecular basis of NTP binding was investigated using a surface plasmon resonance based ligand binding assay and X-ray crystallography. The biophysical and structural data revealed two discrete mechanisms by which MtDTBS binds NTPs: (i) A high affinity binding mode employed by CTP (K-D 160 nM) that is characterized by a slow dissociation rate between enzyme and ligand (k(d) 5.3 X 10(-2) s(-1)) and that is defined by an extended network of specific ligand protein interactions involving both the cytidine and triphosphate moieties and (ii) a low affinity mode employed by the remaining NTPs (K-D > 16.5 mu M), that is characterized by weak interactions between protein and ligand. Previously intractable structures of MtDTBS in complex with ATP, GTP, UTP, and ITP were obtained to define the molecular basis of the low affinity ligand binding. Anchoring of the triphosphate moiety into the phosphate binding loop of MtDTBS allows the promiscuous utilization of multiple NTPs. Both high and low binding mechanisms showed conserved hydrogen bonding interactions involving the beta-phosphate of NTPs and a high-affinity anion binding site within the phosphate binding loop. This study provides insights into enzymes that can likewise utilize multiple NTPs. Refereed/Peer-reviewed

Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of

The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris strain HaA2, can efficiently demethylate 4-methoxybenzoic acid via hemiacetal formation and subsequent elimination of formaldehyde. Oxidative demethylation of a methoxy group para to the carboxyl moiety is strongly favoured over reaction at one in the ortho or meta positions. Dimethoxybenzoic acids containing a para-methoxy group were also efficiently demethylated exclusively at the para position. The presence of additional methoxy substituents reduces the substrate binding affinity and the activity compared to 4-methoxybenzoic acid. The addition of the smaller hydroxy group to the ortho or meta positions or of a nitrogen heteroatom in the aromatic ring of the 4-methoxybenzoate skeleton was better tolerated by the enzyme and these analogues were also readily demethylated. There was no evidence of methylenedioxy ring formation with 3-hydroxy-4-methoxybenzoic acid, an activity which is observed with certain plant CYP enzymes with analogous substrates. CYP199A4 is also able to deprotect the methylenedioxy group of 3,4-(methylenedioxy)benzoic acid to yield 3,4-dihydroxybenzoic acid and formic acid. This study defines the substrate range of CYP199A4 and reveals that substrates without a para substituent are not oxidised with any significant activity. Therefore para-substituted benzoic acids are ideal substrate scaffolds for the CYP199A4 enzyme and will aid in the design of optimised probes to investigate the mechanism of this class of enzymes. They also allow an assessment of the potential of CYP199A4 for synthetic biocatalytic processes involving selective oxidative demethylation or demethenylation.

In Mycobacterium tuberculosis , the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis . From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC 50 ) of 21.4 ± 1 μM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop- R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop- R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor central to fatty acid and glucose homeostasis. PPARγ is the molecular target for type 2 diabetes mellitus (T2DM) therapeutics TZDs (thiazolidinediones), full agonists of PPARγ with robust antidiabetic properties, which are confounded with significant side effects. Partial agonists of PPARγ, such as INT131 (1), have displayed similar insulin-sensitizing efficacy as TZDs, but lack many side effects. To probe the structure-activity relationship (SAR) of the scaffold 1, we synthesized 14 analogs of compound 1 which revealed compounds with higher transcriptional potency for PPARγ and identification of moieties of the scaffold 1 key to high transcriptional potency. The sulfonamide linker is critical to activity, substitutions at position 4 of the benzene ring A were associated with higher transcriptional activity, substitutions at position 2 aided in tighter packing and activity, and the ring type and size of ring A affected the degree of activity.

The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hot-spot’ in SIM1 that is critical for protein function.

There is a real need for simple structures that define a β-strand conformation, a secondary structure that is central to peptide-protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β-strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C-terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X-ray crystallography.

Protein biotinylation is a key post-translational modification found throughout the living world. The covalent attachment of a biotin cofactor onto specific metabolic enzymes is essential for their activity. This modification is distinctive, in that it is carried out by a single enzyme: biotin protein ligase (BPL), an enzyme that is able to biotinylate multiple target substrates without aberrant-off target biotinylation. BPL achieves this target selectivity by recognizing a sequence motif in the context of a highly conserved tertiary structure. One structural class of BPLs has developed an additional ‘substrate verification’ mechanism to further enable appropriate protein selection. This is crucial for the precise and selective biotinylation required for efficient biotin management, especially in organisms that are auxotrophic for biotin. Refereed/Peer-reviewed

The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life-threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding clamp), which clamps the replicative polymerase to DNA. Here we present the first crystal structure of a sliding clamp from a pathogenic fungus (A. fumigatus), at 2.6A. Surprisingly, the structure bears more similarity to the human sliding clamp than other available fungal sliding clamps. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA-interacting protein (PIP-box) motif of human p21 with an affinity (Kd) of 3.1 μm. Molecular dynamics simulations were carried out to better understand how AfumPCNA interacts with human p21. These simulations revealed that the PIP-box bound to AfuPCNA forms a secondary structure similar to that observed in the human complex, with a central 310 helix contacting the hydrophobic surface pocket of AfumPCNA as well as a β-strand that forms an antiparallel sheet with the AfumPCNA surface. Differences in the 310 helix interaction with PCNA, attributed to residue Thr131 of AfumPCNA, and a less stable β-strand formation, attributed to residues Gln123 and His125 of AfumPCNA, are likely causes of the over 10-fold lower affinity of the p21 PIP-box for AfumPCNA as compared to hPCNA. Database The atomic coordinates and structure factors for the Aspergillus fumigatus sliding clamp can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 5TUP.

Summary Peroxisome proliferator-activated receptors (PPARs) are pharmacological targets for the treatment of metabolic disorders. Previously, we demonstrated the anti-diabetic effects of SR1664, a PPARγ modulator lacking classical transcriptional agonism, despite its poor pharmacokinetic properties. Here, we report identification of the antagonist SR11023 as a potent insulin sensitizer with significant plasma exposure following oral administration. To determine the structural mechanism of ligand-dependent antagonism of PPARγ, we employed an integrated approach combining solution-phase biophysical techniques to monitor activation helix (helix 12) conformational dynamics. While informative on receptor dynamics, hydrogen/deuterium exchange mass spectrometry and nuclear magnetic resonance data provide limited information regarding the specific orientations of structural elements. In contrast, label-free quantitative crosslinking mass spectrometry revealed that binding of SR11023 to PPARγ enhances interaction with co-repressor motifs by pushing H12 away from the agonist active conformation toward the H2-H3 loop region (i.e., the omega loop), revealing the molecular mechanism for active antagonism of PPARγ.

Sim1 haploinsufficiency in mice induces hyperphagic obesity and developmental abnormalities of the brain. In humans, abnormalities in chromosome 6q16, a region that includes SIM1, were reported in obese children with a Prader-Willi-like syndrome; however, SIM1 involvement in obesity has never been conclusively demonstrated. Here, SIM1 was sequenced in 44 children with Prader-Willi-like syndrome features, 198 children with severe early-onset obesity, 568 morbidly obese adults, and 383 controls. We identified 4 rare variants (p.I128T, p.Q152E, p.R581G, and p.T714A) in 4 children with Prader-Willi-like syndrome features (including severe obesity) and 4 other rare variants (p.T46R, p.E62K, p.H323Y, and p.D740H) in 7 morbidly obese adults. By assessing the carriers' relatives, we found a significant contribution of SIM1 rare variants to intra-family risk for obesity. We then assessed functional effects of the 8 substitutions on SIM1 transcriptional activities in stable cell lines using luciferase gene reporter assays. Three mutations showed strong loss-of-function effects (p.T46R, p.H323Y, and p.T714A) and were associated with high intra-family risk for obesity, while the variants with mild or no effects on SIM1 activity were not associated with obesity within families. Our genetic and functional studies demonstrate a firm link between SIM1 loss of function and severe obesity associated with, or independent of, Prader-Willi-like features.

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 ( Px-abcc2 ), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD 50 concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella . The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.

Synthetic full agonists of PPARγ have been prescribed for the treatment of diabetes due to their ability to regulate glucose homeostasis and insulin sensitization. While the use of full agonists of PPARγ has been hampered due to severe side effects, partial agonists have shown promise due to their decreased incidence of such side effects in preclinical models. No kinetic information has been forthcoming in regard to the mechanism of full versus partial agonism of PPARγ to date. Here, we describe the discovery of a partial agonist, SR2067. A co-crystal structure obtained at 2.2 Å resolution demonstrates that interactions with the β-sheet are driven exclusively via hydrophobic interactions mediated through a naphthalene group, an observation that is unique from other partial agonists. Surface plasmon resonance revealed that SR2067 binds to the receptor with higher affinity (KD= 513 nM) as compared to that of full agonist rosiglitazone, yet it has a much slower off rate compared to that of rosiglitazone. Refereed/Peer-reviewed

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 Å resolution (Protein Data Bank entry 3U39 ). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.

d -Alanine: d -alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two d -alanine ( d -Ala) molecules to form the d -alanyl: d -alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. d -Cycloserine (DCS), an analog of d -Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and d -alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and d -Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for d -Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC 50 ) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.