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Essential motions in a fungal lipase with bound substrate, covalently attached inhibitor and product

JOURNAL OF MOLECULAR RECOGNITION 2002年第6期15卷 393-404页

作者： Peters, GH Bywater, RP Novo Nordisk AS Biostruct Grp DK-2760 Malov Denmark Tech Univ Denmark Dept Chem MEMPHYS Ctr Biomembrane Phys DK-2800 Lyngby Denmark

As an aid to understanding the influence of dynamic fluctuations during esterolytic catalysis, we follow protein flexibility at three different steps along the catalytic pathway from substrate binding to product clearance via a covalently attached inhibitor, which represents a transition-state mimic. We have applied a classical approach, using molecular dynamics simulations to monitor protein dynamics in the nanosecond regime. We filter out small amplitude fluctuations and focus on the anharmonic contributions to the overall dynamics. This 'essential dynamics' analysis reveals different modes of response along the pathway suggesting that binding, catalysis and product clearance occur along different energy surfaces. Motions in the enzyme with a covalently attached ligand are more complex and occur along several eigenvectors. The magnitudes of the fluctuations in these individual subspaces are significantly smaller than those observed for the substrate and product molecules, indicating that the energy surface is shallow and that a relatively large number of conformational substates are accessible. On the other hand, substrate binding and product release occur at distinct modes of the protein flexibility suggesting that these processes occur along rough energy surfaces with only a few minima. Detailed energetic analyses along the trajectories indicated that in all cases binding is dominated by van der Waals interactions. The carboxylate form of the product is stabilized by a tight hydrogen bond network involving in particular Ser82, which may be a potential cause of product inhibition. Considerations such as these should aid the understanding of mechanisms of substrate, inhibitor or product recognition and could become of importance in the design of new substrates or inhibitors for enzymes. Copyright (C) 2002 John Wiley Sons, Ltd.

关键词： enzyme essential dynamics molecular modelling structure-function relationships protein stability

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Pseudolaric acid analogs as a new class of peroxisome proliferator-activated receptor agonists

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PLANTA MEDICA 2002年第8期68卷 667-671页

作者： Jardat, MS Noonan, DJ Wu, BG Avery, MA Feller, DR Univ Mississippi Dept Pharmacol Sch Pharm University MS 38677 USA Univ Mississippi Natl Ctr Nat Prod Res Sch Pharm University MS 38677 USA Univ Kentucky Coll Med Dept Biochem Lexington KY 40536 USA Univ Mississippi Sch Pharm Dept Med Chem University MS 38677 USA

Extracts of the root and trunk barks of the Chinese tree Pseudolarix kaempferi, which contain pseudolaric acids, are used in Chinese medicine for treatment of fungal infections. Pseudolaric acid B (PLAB) is the major constituent that exhibits anti-fungal activity, The nuclear peroxisome proliferator-activator receptors (PPAR) were proposed as a cellular target for the action of PLAB and its analogs. PLAB and two derivatives were tested for the activation of PPAR isoforms in two mammalian cell lines. CV-I and H4IIEC3 cells were transfected with phorbol ester response element or PPAR response element reporter constructs, and CV-1 cells were co-transfected with the individual PPAR isoform expression plasmids. PLAB showed similar concentration-dependent effects for the activation of PPAR alpha, gamma and delta isoforms in CV-1 and H4IIEC3 cells. O-Deacetylation of PLAB (PLAC) or esterification of the free carboxy group of PLAB with beta-D-O-glucopyranoside (PLAG) markedly reduced or abolished the activation of these PPAR isoforms. In H4IIEC3 cells, PLAB increased the activation of endogenous PPARalpha and the phospholipase C signaling pathway;and stimulated peroxisomal fatty acyl-CoA oxidase activity. These effects of PLAB on the activation of endogenous PPARalpha and phospholipase C-dependent pathway were blocked by staurosporine. These results suggest that the action of PLAB on PPARalpha in H4IIEC3 cells is mediated by a protein kinase C dependent phosphorylation. Based upon these findings, the chemical class of biologically active diterpene acids related to PLAB may have promise for the treatment of metabolic and pathophysiological disorders that are regulated by these nuclear receptor isoforms.

关键词： pseudolaric acids Pseudolarix kaempferi Pinaceae peroxisome proliferator-activated receptors structure-function relationships anti-fungal agents

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Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function

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EUROPEAN JOURNAL OF BIOCHEMISTRY 2002年第24期269卷 6133-6141页

作者： Más, JS Sánchez, CO Ghanem, G Haycock, J Teruel, JAL García-Borrón, JC Jiménez-Cervantes, C Univ Murcia Dept Biochem & Mol Biol Sch Med E-30100 Murcia Spain Free Univ Brussels Brussels Belgium Univ Sheffield Dept Mat Engn Sheffield S Yorkshire England

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R . functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.

关键词： melanocortin 1 receptor melanoma loss-of-function mutations functional coupling structure-function relationships

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Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers

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JOURNAL OF PEPTIDE RESEARCH 2002年第1期60卷 23-36页

作者： Jelokhani-Niaraki, M Prenner, EJ Kay, CM McElhaney, RN Hodges, RS Univ Colorado Hlth Sci Ctr Dept Biochem & Mol Genet Denver CO 80262 USA Wilfrid Laurier Univ Dept Chem Waterloo ON N2L 3C5 Canada Univ Alberta Dept Biochem Edmonton AB T6G 2H7 Canada

To investigate the role of peptide-membrane interactions in the biological activity of cyclic cationic pepticles, the conformations and interactions of four membrane-active antimicrobial pepticles [based on Gramicidin 5 (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidycholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic pepticles include GS10 [Cyclo(VKL (dY) under barP)(2)], GS12 [Cyclo(VKLK (dY) under bar PKVKL (dY) under barP)], GS14 [Cyclo(VKLKV (dY) under bar PLKVKL (dY) under barP)] and [D-Lys](4)GS14 [Cyclo(VKL (dY) under barV (dY) under bar PLKVKL (dY) under barP)] (underlined residues are c)-amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of 3 hemolytic and antimicrobial activities. Interaction of the pepticles with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the pepticles induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [D-Lys](4) GS14>GS14>GS12>GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [D-Lys](4) GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic pepticles with lipid bilayers, the mechanism of their biological activity (and its relation to peptide-lipid interaction) proved to be versatile and complex, and dependent on the biophysical

关键词： biological activity CD spectroscopy cyclic antimicrobial peptides fluorescence spectroscopy gramicidin S analogs peptide-lipid interactions phospholipid vesicles structure-function relationships

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Congenital adrenal hyperplasia due to 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase deficiency

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SEMINARS IN REPRODUCTIVE MEDICINE 2002年第3期20卷 255-276页

作者： Simard, J Moisan, AM Morel, Y Univ Laval Med Ctr Oncol & Mol Endocrinol Res Ctr Canc Genom LabCHULRes Ctr Quebec City PQ G1V 4G2 Canada Univ Lyon INSERM Lab Biochim Endocrinienne U 329 Lyon France Hop Debrousse Lyon France

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. The 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. In humans, there are two 3beta-HSD isoenzymes, which were chronologically designated type I and II encoded by HSD3B1 and HSD3B2 gene, respectively. HSD3B1 gene encodes the almost exclusive 3beta-HSD isoenzyme expressed in the placenta and peripheral tissues, whereas HSD3B2 gene encodes the predominant 3P-HSD isoenzyme expressed in the adrenal gland, ovary, and testis and its deficiency is responsible for a rare form of congenital adrenal hyperplasia causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. Although an elevated ratio of Delta(5)-/Delta(4) -steroids was considered to be the best biological parameter for the diagnosis of this autosomal recessive disorder, the most accurate criteria now appears to be the plasma levels of 17-OH-pregnenolone greater than 100 nmol/L following ACTH stimulation. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing, and 23 missense mutations) have been identified in the HSD3B2 gene in 56 individuals from 44 families suffering from classical 3beta-HSD deficiency. In almost all the cases, the functional characterization of HSD3B2 mutations has provided a molecular explanation for the heterogeneous clinical presentation of this disorder. Indeed these experiments confirm that no functional 3betaHSD type II isoenzyme is expressed in the adrenals and gonads of

关键词： congenital adrenal hyperplasia male pseudohermaphroditism adrenal ovary testis structure-function relationships molecular diagnosis steroidogenesis

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Glycosylation of Thermomyces lanuginosa lipase enhances surface binding towards phospholipids, but does not significantly influence the catalytic activity

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COLLOIDS AND SURFACES B-BIOINTERFACES 2002年第1-2期26卷 125-134页

作者： Peters, GH Svendsen, A Langberg, H Vind, J Patkar, SA Kinnunen, PKJ Tech Univ Denmark Dept Chem Ctr Biomembraen Phys MEMPHYS DK-2800 Lyngby Denmark Novozymes AS DK-2880 Bagsvaerd Denmark Univ Helsinki Inst Biomed Dept Med Chem Helsinki Finland

Binding properties of the native Thermomyces lanuginosa lipase (Tll), the inactive mutant of Tll (S146A;active Ser146 mutated to Ala), and the non-glycosylated mutant of Tll (N33Q) Were determined using fluorescence spectroscopy. Tll, S146A mutant and N33Q mutant show significant different binding behavior to phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, weaker association of lipase molecules is observed to PC liposomes than to PG liposomes. Strong lipase-lipid interactions are observed for the S146A mutant, which is less pronounced for Tll and the N33Q variant. Addition of fatty acid to PG liposomes reduces significantly the binding affinity of the lipases. This effect is less pronounced in fatty acid/PC liposomes. Although the catalytic activity of the N33Q mutant is comparable to Tll, the non-glycosylated variant shows generally lower binding affinity to PC or PG matrix than Tll. Addition of the substrate analog benzene boronic acid (BBA) increases the binding affinity of the S146A and N33Q mutants, while only small changes are observed for Tll suggesting that the dynamics of the active-site lid influences the binding affinity and that the flexibility of the loop region 33-48 might contribute to the activation of the lipase. (C) 2002 Elsevier Science B.V. All rights reserved.

关键词： energy transfer fluorescence spectroscopy interfacial surface quality monolayers structure-function relationships

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The dynamic response of a fungal lipase in the presence of charged surfactants

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COLLOIDS AND SURFACES B-BIOINTERFACES 2002年第1-2期26卷 84-101页

作者： Peters, GH Tech Univ Denmark Dept Chem Ctr Biomembrane Phys MEMPHYS DK-2800 Lyngby Denmark

Lipases are efficient catalysts for lipolytic reactions and require a lipid interface for optimal activity. To study the effect of small charged lipid aggregates on the behavior of these enzymes, we have performed molecular dynamics simulations on five different systems. The simulations carried out in periodic boundary conditions and including explicit water molecules were performed for 2 ns. The lipids were either fatty acid molecules or amines to present negatively or positively charged species. The lipids were partially (i.e. mixture of charged and uncharged lipids) or fully charged. The lipids are randomly placed closely to the active site lid. Our results indicate that in all simulations different lipids do not affect the overall protein structure. However, conformational changes and internal motions in the protein are significantly influenced by the presence of the lipid molecules. In an aqueous environment, protein motions are mainly concentrated in three segments, which are Lys53-Asn63, Ser83-Asn86 and the C-terminus. The former region is structurally conserved in the lipase family and has been proposed to be involved in the activation of lipases. Interestingly, in the presence of the lipid molecules these fluctuations are suppressed, which is partly due to the interaction of the C-terminus with the lipid molecules. This may suggest that the C-terminus (which is not conserved in the lipase family) may have an important role in recognizing and binding to different lipid surfaces. In the presence of lipid aggregates, fluctuations are observed in the loops Va197-Lys106, Arg202-Gly214 and Trp223-Thr252. In particular, the flexibility of the loop Va197-Lys106 may have fundamental implications for the activation of lipases. This region has been suggested as a possible 'entering gate' for substrate molecules to the binding cleft. (C) 2002 Elsevier Science B.V. All rights reserved.

关键词： lipid patch molecular dynamics simulations molecular modeling protein flexibility Rhizomucor miehei lipase structure-function relationships

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The dynamic response of a fungal lipase in the presence of charged surfactants

The dynamic response of a fungal lipase in the presence of c...

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European Meeting on Lipid and Lipase Interaction

作者： Peters, GH Tech Univ Denmark Dept Chem Ctr Biomembrane Phys MEMPHYS DK-2800 Lyngby Denmark

关键词： lipid patch molecular dynamics simulations molecular modeling protein flexibility Rhizomucor miehei lipase structure-function relationships

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Molecular design versus empirical discovery in peptide-based vaccines. Coming to terms with fuzzy recognition sites and ill-defined structure-function relationships in immunology

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VACCINE 1999年第3-4期18卷 216-221页

作者： van Regenmortel, MHV CNRS Inst Biol Mol & Cellulaire F-67084 Strasbourg France

In view of our increased understanding of the molecular basis of immunological recognition, it is commonly believed that it should be possible to apply molecular design strategies to the development of peptide-based vaccines. The stated aim is to transform the development of a Vaccine from a trial and error empirical operation into a so-called rational, structure-based process. In the present review, it is argued that it is misleading to oppose rational and empirical approaches in vaccine research since both are needed in the practice of experimental science. Many reasons are given for the view-that the molecular design of synthetic vaccines is not a realistic scientific enterprise. The capacity of a peptide to induce a protective immune response depends on many extrinsic factors and regulatory mechanisms of the recipient host which are not amenable to molecular design of the peptide immunogen. It seems safe to predict that the development of peptide-based vaccines will continue to be driven by empirical discovery rather than by so-called rational design, (C) 1999 Elsevier Science Ltd. AU rights reserved.

关键词： molecular design peptide-based vaccines structure-function relationships

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Glycosylation of Thermomyces lanuginosa lipase enhances surface binding towards phospholipids, but does not significantly influence the catalytic activity

Glycosylation of <i>Thermomyces lanuginosa</i> lipase enhanc...

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European Meeting on Lipid and Lipase Interaction

关键词： energy transfer fluorescence spectroscopy interfacial surface quality monolayers structure-function relationships

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