Molecular Simulation Elaborating the Mechanism of 1?-Hydroxy Alantolactone Inhibiting Ubiquitin-Conjugating Enzyme UbcH5s.
ABSTRACT: 1?-hydroxy alantolactone, a sesquiterpene lactone, exhibits potent anti-inflammatory and anticancer activities. Recently, it has been found to target UbcH5s by covalently bonding with Cys85 specifically, but the exact molecular basis remains unclear. Here, we analyzed the structural specificity of the catalytic site of UbcH5s by comparing them with other E2 proteins. Molecular dynamics was performed to detect the structural stability of the catalytic site. Docking method was then used to predict conformations of ligand docked at the catalytic site of UbcH5s. The electrostatic surface and charge distribution of ligand and proteins were analyzed by quantitative calculation. Molecular dynamics was used to detect the stability of docking complexes of 1?-hydroxy alantolactone and UbcH5s, the covalently bonded intermediates and the products. The QM/MM methodology was used to calculate the free energy barrier of hydrogen transfer and formation of covalent bond between 15-position carbon of ligand and Cys85. Results revealed that the structure of the catalytic site is stable, and 1?-hydroxy alantolactone can dock at the catalytic site with correct conformation. Molecular dynamics further demonstrates that 1?-hydroxy alantolactone can steadily combine with UbcH5s. Intermediate and product of catalytic reaction are also certified to be stable. Besides, Asp112 and Asn114 function as anchors to fix ligand, ensuring it steadily docked at catalytic site to complete covalent reaction. More importantly, we have found that Cys85 of UbcH5c is more efficient to form a covalent bond with the ligand in comparison with UbcH5a and UbcH5b. Our results successfully explained the mechanism of 1?-hydroxy alantolactone covalently bonding with UbcH5s. Such molecular mechanism may provide a better insight into the molecular development or modification for ubiquitin-related drugs.
Project description:GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPAR?). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPAR? ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPAR? LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists. Here, we developed a series of analogs based on the orthosteric covalent antagonist scaffold with the goal of inhibiting both orthosteric and allosteric cellular activation of PPAR? by MRL20, an orthosteric agonist that also binds to an allosteric site. Our efforts resulted in the identification of SR16832 (compound 22), which functions as a dual-site covalent inhibitor of PPAR? transcription by PPAR?-binding ligands. Molecular modeling, protein NMR spectroscopy structural analysis, and biochemical assays indicate the inhibition of allosteric activation occurs in part through expansion of the 2-chloro-5-nitrobenzamidyl orthosteric covalent antagonist toward the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPAR? activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPAR? when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPAR? ligands such as docosahexaenoic acid (DHA) compared to orthosteric covalent antagonists. Compounds such as SR16832 may be useful chemical tools to use as a dual-site bitopic orthosteric and allosteric covalent inhibitor of ligand binding to PPAR?.
Project description:Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.
Project description:1?-hydroxy alantolactone, a sesquiterpene lactone mainly isolated from Inula genus plants, exhibits potent anti-inflammatory and anticancer activities. In this work, 1?-hydroxy alantolactone was isolated and five derivatives were prepared through different reactions at the C1-OH and C13-methylene motifs. The structure-activity relationships (SAR) of anti-inflammatory effects against NO production in RAW264.7 cells showed that the ?-methylene-?-butyrolactone motif was essential for NO production suppression and that retaining the C1-OH group can remarkably improve this effect. The NF-?B signaling pathway plays a pivotal role in the regulation of NO expression. Moreover, the levels of p65 and p50 phosphorylation were investigated and active compound 1 inhibited phosphorylation of p65 and p50 in TNF-?-induced NF-?B signaling. Further molecular docking suggested that 1 may target the p65 of NF-?B.
Project description:Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs(3+)) binds to three cysteine residues, methylarsenite (MMA(3+)) binds to two, and dimethylarsenite (DMA(3+)) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs(3+) and active in the methylation of MMA(3+). The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S(C32) is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S(C61) and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S(C61) and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.
Project description:Covalently bound protein kinase inhibitors have been frequently designed to target noncatalytic cysteines at the ATP binding site. Thus, it is important to know if a given cysteine can form a covalent bond. Here we combine a function-site interaction fingerprint method and DFT calculations to determine the potential of cysteines to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site cysteine data set was assembled. The orientation of the cysteine thiol group indicates which cysteines can potentially form covalent bonds. These covalent inhibitor easy-available cysteines are located within five regions: P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop, and DFG-3 close to the DFG peptide. In an independent test set these cysteines covered 95% of covalent kinase inhibitors. This study provides new insights into cysteine reactivity and preference which is important for the prospective development of covalent kinase inhibitors.
Project description:Sesquiterpene lactones (STLs) are a class of plant secondary metabolites widely found in nature with potent antitumor activities. In this work, two isolated STLs 1β-hydroxy alantolactone (1) and ivangustin (2) were derivatized through diversity-oriented strategy, and in vitro cytotoxic activity assessments were conducted against six cell lines including HeLa, PC-3, HEp-2, HepG2, CHO and HUVEC. The cytotoxic structure-activity relationship showed that the double bond between C5 and C6 was beneficial to improve activity; C1-OH oxidized derivatives showed a slight stronger activity, comparable to the positive drug etoposide (VP-16). Yet, C1-OH esterified derivatives decreased the potency which were different from those of 1-O-acetylbritannilactone (ABL) reported previously by us, and C13-methylene reductive and spiro derivatives resulted in almost complete ablation of cytotoxic activity. Mechanistic basis of cytotoxicity of the representative compound 1i was assayed to relate with apoptosis and cell cycle arrest. Furthermore, 1i inhibited TNF-α-induced canonical NF-κB signaling in PC-3 cells. Molecular modeling studies exhibited additional hydrogen bond interaction between 1i and the residue Lys37 of p65, indicating that 1i could form covalent protein adducts with Cys38 on p65.
Project description:The chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. The soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6??s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25??s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione-binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. The results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled.
Project description:DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.
Project description:We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity.
Project description:The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.