The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.
A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.
Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.
The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.
The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.
HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligandbinding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU-486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU-486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist-bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist-bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist-bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist-bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor. Keywords: glucocorticoid receptor; H/D exchange; mass spectrometrySupplemental material: see www.proteinscience.orgThe glucocorticoid receptor (GR) is a member of the nuclear receptor family; its members include the mineralcorticoid (MR), progesterone (PR), estrogen (ER), and androgen (AR) receptors (Robinson-Rechavi et al. 2001). The glucocorticoid receptor has been shown to regulate glucose homeostasis, lipid metabolism, and inflammation (Reichardt et al. 2000), making it a therapeutically important drug target. Marketed steroids such as dexamethasone and prednisolone are effective at suppressing inflammation but also show negative side effects, including diabetes, hyperglycemia, and osteoporosis (Schacke et al. 2002). Pharmaceutical research continues to seek high affinity glucocorticoid receptor ligands that have potent anti-inflammatory activity with reduced side effects. Much work has been done to identify novel inhibitors that show such dissociative behavior (Coghlan et al. 2003;Schacke et al. 2004).GR is a transcription factor that is activated upon ligand binding. Upon glucocorticoid binding, the GR receptor translocates from the cytoplasm into the nucleus, where it serves either directly or indirectly as a transcriptional regulator. Transrepression of genes is understood to be Reprint requests to: Lee Frego, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877, USA; e-mail: lfrego@ rdg.boehringer-ingelheim.com; fax: (203) 791-6196.Abbreviations: H/D, hydrogen/deuterium; HXMS, hydrogen/deuterium exchange mass spectrometry; LC/MS, liquid chromatography/mass spectrometry; GR, glucocorticoid receptor; GR LBD, glucocorticoid receptor ligand-binding domain; TIF2, transcriptional intermediary factor 2; SRC1, steroid receptor coactivator 1; NCoR, nuclear receptor corepressor; NF-kB, nuclear factor kB; TAT, tyrosine aminotransferase; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; DBD, DNA-binding domain; LBD, ligand-binding domain; NMR, nuclear magnetic resonance.Article and publication are at
Recent reports have demonstrated the potential of monocyclic β-lactam derivatives as inhibitors of human cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and mass spectrometry has revealed the presence of an acylenzyme intermediate suggesting that β-lactams are hydrolyzed by the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic β-lactam derivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4-methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylation of the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of the hydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuable information on acylation and deacylation rates which relate to the IC50 values observed for β-lactams. More importantly the potential of compound 1 as an active site titrating agent for HCMV protease has been exploited, and a simple protocol for rapid determination of active enzyme is described. The data are consistent with the HCMV protease dimer being composed of two functional active sites. This titrating agent represents an important tool that should significantly facilitate the characterization of this novel enzyme.
Electron-capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) employed to generate comprehensive sequence information for the chromatographic analysis of enzymatic protein digests is described. A pepsin digest of cytochrome c was separated by reversed-phase micro-high-performance liquid chromatography (microHPLC) and ionized 'on-line' by electrospray ionization (ESI). The ions thus formed were transferred to and trapped in the FTMS analyzer cell. Typically, no precursor ion isolation was performed. The trapped ions were subjected to a pulse of electrons to induce fragmentation. Mass spectra were acquired continuously to produce a three-dimensional LC/MS data set. The spectra were dominated by c and, to a lesser degree, z ions, which provided near complete sequence coverage. External calibration provided good mass accuracy and resolution, typical of FTMS. Thus microHPLC/ECD - FTMS is shown to be a highly informative method for the analysis of enzymatic protein digests.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.