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1.
Biochemistry ; 60(17): 1327-1336, 2021 05 04.
Article in English | MEDLINE | ID: mdl-33724805

ABSTRACT

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisX and GalNAc, respectively.


Subject(s)
Acetylgalactosamine/metabolism , Antigens, Tumor-Associated, Carbohydrate/metabolism , Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Animals , Crystallography, X-Ray , Humans , Ligands , Mice , Protein Binding , Protein Domains
2.
Biochim Biophys Acta Gen Subj ; 1865(2): 129800, 2021 02.
Article in English | MEDLINE | ID: mdl-33246032

ABSTRACT

BACKGROUND: Due to the importance of both prostaglandins (PGs) and leukotrienes (LTs) as pro-inflammatory mediators, and the potential for eicosanoid shunting in the presence of pathway target inhibitors, we have investigated an approach to inhibiting the formation of both PGs and LTs as part of a multi-targeted drug discovery effort. METHODS: We generated ligand-protein X-ray crystal structures of known inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) and the 5-Lipoxygenase Activating Protein (FLAP), with their respective proteins, to understand the overlapping pharmacophores. We subsequently used molecular modeling and structure-based drug design (SBDD) to identify hybrid structures intended to inhibit both targets. RESULTS: This work enabled the preparation of compounds 4 and 5, which showed potent in vitro inhibition of both targets. SIGNIFICANCE: Our findings enhance the structural understanding of mPGES-1 and FLAP's unique ligand binding pockets and should accelerate the discovery of additional dual inhibitors for these two important integral membrane protein drug targets.


Subject(s)
5-Lipoxygenase-Activating Protein Inhibitors/pharmacology , Drug Discovery , Eicosanoids/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Prostaglandin-E Synthases/antagonists & inhibitors , 5-Lipoxygenase-Activating Protein Inhibitors/chemistry , 5-Lipoxygenase-Activating Proteins/metabolism , Eicosanoids/metabolism , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Prostaglandin-E Synthases/metabolism , Structure-Activity Relationship
3.
J Lipid Res ; 61(10): 1347-1359, 2020 10.
Article in English | MEDLINE | ID: mdl-32690595

ABSTRACT

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Lipoprotein Lipase/chemistry , Lipoprotein Lipase/immunology , Animals , Binding Sites , Humans , Mice , Tryptophan
4.
Bioorg Med Chem Lett ; 27(6): 1478-1483, 2017 03 15.
Article in English | MEDLINE | ID: mdl-28190634

ABSTRACT

We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC80 of 24nM for inhibition of PGE2 formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC80 in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.


Subject(s)
Benzoates/chemistry , Benzoates/pharmacology , Drug Discovery , Microsomes/drug effects , Prostaglandin-E Synthases/antagonists & inhibitors , Animals , Crystallography, X-Ray , Dogs , Microsomes/enzymology , Prostaglandin-E Synthases/chemistry , Rats
5.
Bioorg Med Chem Lett ; 26(19): 4824-4828, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27554445

ABSTRACT

Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE2 synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7nM. In addition, 14 had no activity in human COX-1 or COX-2 assays at 30µM, and failed to inhibit human mPGES-2 at 62.5µM in a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction of PGE2. In dog, 14 had oral bioavailability (74%), clearance (3.62mL/(min*kg)) and volume of distribution (Vd,ss=1.6L/kg) values within our target ranges. For these reasons, 14 was selected for further study.


Subject(s)
Piperidines/chemistry , Piperidines/pharmacology , Prostaglandin-E Synthases/antagonists & inhibitors , A549 Cells , Animals , Crystallography, X-Ray , Dogs , Humans , Piperidines/pharmacokinetics , Rats , Species Specificity , Structure-Activity Relationship
7.
J Med Chem ; 59(1): 194-205, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26653180

ABSTRACT

As part of a program aimed at the discovery of antinociceptive therapy for inflammatory conditions, a screening hit was found to inhibit microsomal prostaglandin E synthase-1 (mPGES-1) with an IC50 of 17.4 µM. Structural information was used to improve enzyme potency by over 1000-fold. Addition of an appropriate substituent alleviated time-dependent cytochrome P450 3A4 (CYP3A4) inhibition. Further structure-activity relationship (SAR) studies led to 8, which had desirable potency (IC50 = 12 nM in an ex vivo human whole blood (HWB) assay) and absorption, distribution, metabolism, and excretion (ADME) properties. Studies on the formulation of 8 identified 8·H3PO4 as suitable for clinical development. Omission of a lipophilic portion of the compound led to 26, a readily orally bioavailable inhibitor with potency in HWB comparable to celecoxib. Furthermore, 26 was selective for mPGES-1 inhibition versus other mechanisms in the prostanoid pathway. These factors led to the selection of 26 as a second clinical candidate.


Subject(s)
Analgesics/chemical synthesis , Analgesics/pharmacology , Cyclooxygenase Inhibitors/chemical synthesis , Cyclooxygenase Inhibitors/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Intramolecular Oxidoreductases/antagonists & inhibitors , Microsomes/enzymology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Biological Availability , Celecoxib/pharmacology , Cyclooxygenase Inhibitors/pharmacokinetics , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Dogs , Drug Discovery , Humans , Microsomes/drug effects , Models, Molecular , Prostaglandin-E Synthases , Rats , Structure-Activity Relationship
8.
J Med Chem ; 59(2): 750-5, 2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26683992

ABSTRACT

A transdermal SARM has a potential to have therapeutic benefit through anabolic activity in muscle while sparing undesired effects of benign prostate hyperplasia (BPH) and liver-mediated decrease in HDL-C. 2-Chloro-4-[(2-hydroxy-2-methyl-cyclopentyl)amino]-3-methyl-benzonitrile 6 showed the desired muscle and prostate effects in a preclinical ORX rat model. Compound 6 had minimal effect on HDL-C levels in cynomolgus monkeys and showed human cadaver skin permeability, thus making it an effective tool for proof-of-concept studies in a clinical setting.


Subject(s)
Anabolic Agents/therapeutic use , Androgen Antagonists/therapeutic use , Aniline Compounds/therapeutic use , Muscular Atrophy/drug therapy , Nitriles/therapeutic use , Administration, Cutaneous , Anabolic Agents/administration & dosage , Anabolic Agents/chemical synthesis , Androgen Antagonists/administration & dosage , Androgen Antagonists/chemical synthesis , Aniline Compounds/administration & dosage , Aniline Compounds/chemical synthesis , Animals , Cholesterol, HDL/metabolism , Humans , Hypercholesterolemia/chemically induced , In Vitro Techniques , Liver/drug effects , Liver/metabolism , Macaca fascicularis , Male , Models, Molecular , Nitriles/administration & dosage , Nitriles/chemical synthesis , Orchiectomy , Prostatic Hyperplasia/chemically induced , Rats , Skin Absorption , Structure-Activity Relationship
9.
J Med Chem ; 58(16): 6607-18, 2015 Aug 27.
Article in English | MEDLINE | ID: mdl-26218343

ABSTRACT

To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts.


Subject(s)
Dyslipidemias/drug therapy , Receptors, Glucocorticoid/agonists , Receptors, Glucocorticoid/antagonists & inhibitors , Animals , Binding Sites , Cricetinae , Crystallography, X-Ray , Diet, High-Fat , Female , Ligands , Lipids/blood , Mesocricetus , Models, Molecular , Protein Conformation , Rats , Rats, Wistar , Receptors, Glucocorticoid/genetics , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology
10.
J Med Chem ; 58(11): 4727-37, 2015 Jun 11.
Article in English | MEDLINE | ID: mdl-25961169

ABSTRACT

Microsomal prostaglandin E synthase 1 (mPGES-1) is an α-helical homotrimeric integral membrane inducible enzyme that catalyzes the formation of prostaglandin E2 (PGE2) from prostaglandin H2 (PGH2). Inhibition of mPGES-1 has been proposed as a therapeutic strategy for the treatment of pain, inflammation, and some cancers. Interest in mPGES-1 inhibition can, in part, be attributed to the potential circumvention of cardiovascular risks associated with anti-inflammatory cyclooxygenase 2 inhibitors (coxibs) by targeting the prostaglandin pathway downstream of PGH2 synthesis and avoiding suppression of antithrombotic prostacyclin production. We determined the crystal structure of mPGES-1 bound to four potent inhibitors in order to understand their structure-activity relationships and provide a framework for the rational design of improved molecules. In addition, we developed a light-scattering-based thermal stability assay to identify molecules for crystallographic studies.


Subject(s)
Analgesics/chemistry , Anti-Inflammatory Agents/chemistry , Drug Design , Enzyme Inhibitors/chemistry , Imidazoles/chemistry , Intramolecular Oxidoreductases/chemistry , Amino Acid Sequence , Analgesics/metabolism , Analgesics/therapeutic use , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/therapeutic use , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Humans , Intramolecular Oxidoreductases/metabolism , Microsomes/enzymology , Models, Molecular , Molecular Sequence Data , Molecular Structure , Prostaglandin-E Synthases , Protein Conformation , Sequence Homology, Amino Acid
11.
J Med Chem ; 57(3): 849-60, 2014 Feb 13.
Article in English | MEDLINE | ID: mdl-24446728

ABSTRACT

The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path for further insights into structure-based ligand design.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Benzoxepins/chemistry , Receptors, Glucocorticoid/chemistry , Sulfonamides/chemistry , Acute Disease , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/chemically induced , Arthritis, Experimental/drug therapy , Benzoxepins/pharmacokinetics , Benzoxepins/pharmacology , Binding Sites , Carrageenan , Cell Line , Chronic Disease , Collagen , Crystallography, X-Ray , Drug Design , Edema/chemically induced , Edema/drug therapy , Humans , Inflammation/drug therapy , Ligands , Male , Models, Molecular , Molecular Conformation , Rats , Rats, Sprague-Dawley , Receptors, Glucocorticoid/metabolism , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology
12.
PLoS One ; 8(12): e84147, 2013.
Article in English | MEDLINE | ID: mdl-24367637

ABSTRACT

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.


Subject(s)
Catalytic Domain/genetics , Disease/genetics , Mutation , Polycomb Repressive Complex 2/chemistry , Polycomb Repressive Complex 2/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , Enhancer of Zeste Homolog 2 Protein , Humans , Models, Molecular , Molecular Sequence Data , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/genetics , Sf9 Cells , Spodoptera
13.
J Med Chem ; 56(3): 963-9, 2013 Feb 14.
Article in English | MEDLINE | ID: mdl-23311358

ABSTRACT

The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.


Subject(s)
Carbazoles/pharmacology , Histone Deacetylase Inhibitors/pharmacology , NAD/metabolism , Sirtuin 1/metabolism , Carbazoles/chemistry , Catalytic Domain , Crystallography, X-Ray , Histone Deacetylase Inhibitors/chemistry , Humans , Models, Molecular , Protein Conformation , Sirtuin 1/chemistry , Surface Plasmon Resonance
14.
J Mol Biol ; 397(4): 883-92, 2010 Apr 09.
Article in English | MEDLINE | ID: mdl-20156452

ABSTRACT

PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel beta sandwich fold composed of 11 antiparallel beta-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092-->Glu, observed in the Caenorhabditis elegans ortholog RPM-1.


Subject(s)
Amino Acid Substitution/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/chemistry , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans , Crystallography, X-Ray , Mice , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Alignment , Ubiquitin-Protein Ligases
15.
Nat Methods ; 5(2): 135-46, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18235434

ABSTRACT

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.


Subject(s)
Chemical Fractionation/methods , Chemistry, Physical/methods , Protein Engineering/methods , Proteomics/methods , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
16.
J Mol Biol ; 352(5): 1019-28, 2005 Oct 07.
Article in English | MEDLINE | ID: mdl-16140327

ABSTRACT

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.


Subject(s)
Herpesvirus 8, Human/chemistry , Macrophage Inflammatory Proteins/chemistry , Sarcoma, Kaposi/virology , Amino Acid Sequence , Chemokine CCL4 , Crystallography, X-Ray , Glycosaminoglycans/metabolism , Herpesvirus 8, Human/genetics , Humans , Macrophage Inflammatory Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Receptors, CCR8 , Receptors, Chemokine/metabolism , Static Electricity
17.
J Mol Biol ; 343(4): 1019-34, 2004 Oct 29.
Article in English | MEDLINE | ID: mdl-15476818

ABSTRACT

The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.


Subject(s)
Aldehyde-Lyases/chemistry , Ribosemonophosphates/metabolism , Aldehyde-Lyases/metabolism , Antibodies/chemistry , Antibodies/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Dimerization , Escherichia coli/enzymology , Protein Structure, Tertiary , Static Electricity
18.
Curr Biol ; 14(8): 718-24, 2004 Apr 20.
Article in English | MEDLINE | ID: mdl-15084288

ABSTRACT

Structural studies of cellular immune receptors such as MHC molecules, T cell receptors (TCR), and TCR/MHC complexes have been carried out with recombinant, soluble forms of the extracytoplasmic domain of these glycoproteins. The important role of the membrane bilayer in T cell recognition and antigen presentation has become increasingly obvious with the description of lipid microdomains. These rafts appear to regulate recognition and signaling by clustering receptors and facilitating the formation of the immune synapse. However, the interactions and orientation of these receptors at the lipid bilayer are unknown. We have used H-2K(b), a major-histocompatibility (MHC) class I molecule, and tethered its soluble domain to a lipid bilayer via a surrogate connecting peptide to reveal the disposition of MHC molecule on the membrane surface. We demonstrate that the long axis of the MHC molecule is approximately parallel to the plane of the membrane with the peptide binding pocket close to the membrane surface. This result was determined by analyzing 4.5A resolution electron crystallographic projection data from frozen-hydrated 2-dimensional crystals. Ionic interactions between the lipid headgroup and the protein appear to be responsible for this orientation, which could establish a "fourth dimension" during MHC/T cell receptor interactions critical for activation.


Subject(s)
Antigen-Antibody Complex/metabolism , Cell Membrane/metabolism , H-2 Antigens/metabolism , Lipid Bilayers/metabolism , T-Lymphocytes/metabolism , Animals , CD8 Antigens/metabolism , Cell Membrane/immunology , Crystallography , H-2 Antigens/immunology , Mice , Models, Molecular , Protein Conformation , Static Electricity , Surface Plasmon Resonance , T-Lymphocytes/immunology
19.
J Immunol ; 172(5): 2994-3002, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14978103

ABSTRACT

We identify and consider some characteristics of a peptide antagonist for the Ag-specific receptor on 2C cells (the 2C TCR). The peptide, GNYSFYAL (called GNY), binds to H-2K(b), and a very high-resolution crystal structure of the GNY-K(b) complex at 1.35 A is described. Although the GNY peptide does not bind to L(d), the potency of GNY-K(b) as an antagonist is evident from its ability to specifically inhibit 2C TCR-mediated reactions to an allogenic agonist complex (QLSPFPFDL-L(d)), as well as to a syngeneic agonist complex (SIYRYYGL-K(b)). The crystal structure and the activities of alanine-substituted peptide variants point to the properties of the peptide P4 side chain and the conformation of the Tyr-P6 side chain as the structural determinants of GNYSFYAL antagonist activity.


Subject(s)
Isoantigens/physiology , Oligopeptides/chemistry , Oligopeptides/physiology , Receptors, Antigen, T-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/physiology , Alanine/metabolism , Amino Acid Sequence , Amino Acid Substitution/immunology , Animals , Arginine/metabolism , Cell Line , Cell Line, Tumor , Clone Cells , Crystallography, X-Ray , Cytotoxicity Tests, Immunologic , Female , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Lysine/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligopeptides/metabolism , Protein Binding/immunology , Receptors, Antigen, T-Cell/agonists , Serine/metabolism , Structure-Activity Relationship
20.
Genes Dev ; 17(8): 977-90, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12672694

ABSTRACT

In Caenorhabditis elegans, an X chromosome-counting mechanism specifies sexual fate. Specific genes termed X-signal elements, which are present on the X chromosome, act in a concerted dose-dependent fashion to regulate levels of the developmental switch gene xol-1. In turn, xol-1 levels determine sexual fate and the activation state of the dosage compensation mechanism. The crystal structure of the XOL-1 protein at 1.55 A resolution unexpectedly reveals that xol-1 encodes a GHMP kinase family member, despite sequence identity of 10% or less. Because GHMP kinases, thus far, have only been characterized as small molecule kinases involved in metabolic pathways, for example, amino acid and cholesterol synthesis, XOL-1 is the first member that controls nonmetabolic processes. Biochemical investigations demonstrated that XOL-1 does not bind ATP under standard conditions, suggesting that XOL-1 acts by a mechanism distinct from that of other GHMP kinases. In addition, we have cloned a XOL-1 ortholog from Caenorhabditis briggsae, a related nematode that diverged from C. elegans approximately 50-100 million years ago. These findings demonstrate an unanticipated role for GHMP kinase family members as mediators of sexual differentiation and dosage compensation and, possibly, other aspects of differentiation and development.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Caenorhabditis/genetics , Dosage Compensation, Genetic , Helminth Proteins/chemistry , Helminth Proteins/physiology , Sex Determination Processes , X Chromosome , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Caenorhabditis/growth & development , Caenorhabditis/metabolism , Caenorhabditis elegans/growth & development , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Disorders of Sex Development/genetics , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Spectrometry, Fluorescence
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