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L Moroder

Publications and source records attributed to L Moroder.

At least 55 records · Page 3Linked to original sources
PubMed1999

Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site interact with the penultimate aspartic acid and the C-terminal amide of cholecystokinin.

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR.CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.

Amides↗
PubMed1999

Bivalency as a principle for proteasome inhibition.

The proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern. This lack of well-defined substrate specificities makes the design of peptide-based highly selective inhibitors extremely difficult. However, the x-ray structure of the proteasome from Saccharomyces cerevisiae reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Structure-derived active site separation distances were exploited for the design of homo- and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a flexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber. Spacer lengths were selected that satisfy the inter- and intra-ring distances for occupation of the active sites from the S subsites. X-ray analysis of the proteasome/bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups. Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding. The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes. The present study confirms its utility also for inhibition of multicatalytic protease complexes.

Aldehydes↗
PubMed1999

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single-stranded collagen type I peptides related to the collagenase cleavage site.

Synthetic collagen peptides containing larger numbers of Gly-Pro-Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen-type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc-Gly-Pro-Hyp(tBu)-OH and Fmoc-Pro-Hyp-Gly-OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc-Gly-Pro-Hyp(tBu)-OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly-Pro units from the growing peptide chain. Conversely, by the use of the Fmoc-Pro-Hyp-Gly-OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen-type peptides were obtained in satisfactory yields as highly homogeneous compounds.

Amino Acid Sequence↗
PubMed1999

Benzotriazonine as a new core structure for the design of CCK-receptor antagonists.

The search for heterocyclic scaffolds for the design of non-peptidic and highly selective agonists or antagonists of peptide hormone receptors led to 4-N-benzyl-2,3,4,5,6,7-hexahydro-1H-1,4,7-benzotriazonin-2, 6-dione with a 9-membered core structure as a new low mass lead compound that exhibits submicromolar antagonistic activity at the CCK-A receptor with a 54-fold selectivity over the CCK-B/gastrin receptor.

Animals↗
PubMed1999

Photomodulation of conformational states. Synthesis of cyclic peptides with backbone-azobenzene moieties.

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4-aminomethyl)phenylazobenzoic acid (H-AMPB-OH) and (4-amino)phenylazobenzoic acid (H-APB-OH) were synthesized and used to cyclize a bis-cysteinyl-octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H-AMPB-OH proceeded smoothly on a chlorotrityl-resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H-APB-OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S-tert-butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S-trityl protection. Side-chain cyclization of the APB-peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side-chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly.

Azo Compounds↗
PubMed1999

Exogenous CCK and gastrin stimulate pancreatic exocrine secretion via CCK-A but also via CCK-B/gastrin receptors in the calf.

A predominance of the pancreatic cholecystokinin (CCK) receptor of the B/gastrin subtype (CCK-B/G) was reported in calves older than 1 month. Specific CCK-A and CCK-B/G receptor antagonists (SR 27897 and PD 135158, respectively) were used to identify the CCK receptor subtype involved in exogenous CCK- and gastrin-induced exocrine pancreatic responses. Conscious calves (2 months old) with catheterized pancreas, jugular vein and duodenum were used; the pancreatic juice was continuously reinfused. CCK (30 pmol kg-1 min-1, 40 min) evoked an increase in pancreatic juice flow and enzyme secretion, while the same dose of gastrin increased enzyme secretion alone. CCK-induced pancreatic secretion was abolished by SR 27897 (15 nmol kg-1 min-1, 55 min) and reduced by PD 135158 (0.15 nmol kg-1 min-1, 55 min). Gastrin-induced enzyme secretion was reduced by PD 135158 (50% to 90%) and to a lesser extent by SR 27897 (50% to 60%). These results demonstrate that CCK and gastrin in the physiological range stimulate pancreatic exocrine secretion in calves and that these effects are partly mediated by CCK-B/G receptors. Although CCK-A receptors are not predominantly expressed, they seem to play a major role in the response of pancreatic exocrine secretion to CCK.

Animals↗
PubMed1999

Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes.

BACKGROUND: The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action. Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. RESULTS: The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. CONCLUSIONS: Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.

Binding Sites↗
PubMed1999

Toward the experimental codon reassignment in vivo: protein building with an expanded amino acid repertoire.

The high precision and fidelity of the genetic message transmission are ensured by numerous proofreading steps, from DNA replication and transcription to protein translation. The key event for translational fidelity is the proper codon assignment for 20 canonical amino acids. An experimental codon reassignment is possible for noncanonical amino acids in vivo using artificially constructed expression hosts under efficient selective pressure. However, such amino acids may interfere with the cellular metabolism and thus do not belong to the 'first' or 'restricted' part of the universal code, but rather to a second or 'relaxed' part, which is limited mainly by the downstream proofreading in the natural translational machinery. Correspondingly, not all possible alpha-amino acids can be introduced into proteins. The aim of this study is to discuss biological and evolutionary constraints on possible candidates for this second coding level of the universal code. Engineering of such a 'second' code is expected to have great academic as well as practical impact, ranging from protein folding studies to biomedicine.

Acylation↗
PubMed1999

Arginine 197 of the cholecystokinin-A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin.

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.

Amino Acid Sequence↗
PubMed1999

The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized alpha-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.

Amino Acid Sequence↗
PubMed1998

Isomorphous replacement of cystine with selenocystine in endothelin: oxidative refolding, biological and conformational properties of [Sec3,Sec11,Nle7]-endothelin-1.

Air re-oxidation of fully reduced human endothelin-1 under optimized conditions yields the natural isomer with parallel disulfide bridges and the non-natural isomer with crossed disulfide bridges at a ratio of 3:1. In view of the recently determined highly reducing redox potential of selenocysteine (-381 mV) in peptides, the half-cystine residues Cys3 and Cys11 of the natural isomer of endothelin-1 were replaced by selenocysteine. Taking advantage of the high stability of the diselenide group toward reducing agents for disulfides a regioselective disulfide bridging of the second cysteine pair allowed for straightforward preparation of the [Sec3,Sec11, Nle7]-endothelin-1. NMR structural analysis showed conformational preferences of this endothelin analog that were identical to those of the natural hormone. Similarly, the bioactivity data confirmed that replacement of cysteine residues with selenocysteine was without detectable effect on receptor recognition and signal transduction. Both findings strongly support that the exchange of sulfur against selenium produces a fully isomorphous molecule as recently observed for similar exchanges at the level of methionine residues in proteins. Moreover, oxidative refolding of the fully reduced [Sec3,Sec11,Nle7]-endothelin-1 fulfilled the expectation that the redox potential of the selenocysteines would dictate quantitative formation of the natural isomer. These results suggest that the selenocysteine approach, besides offering an interesting chemical tool for induction of correct oxidative folding of multiple cysteine-containing peptides, should even allow for the preparation of non-natural isomers and thus for studying conformational preferences of folding intermediates in peptides and proteins.

Amino Acid Sequence↗
PubMed1998

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

Animals↗
PubMed1998

Bis-substituted malonic acid hydroxamate derivatives as inhibitors of human neutrophil collagenase (MMP8).

Malonic acid hydroxamate derivatives bis-substituted at the methylene group were synthesized as potential nonpeptidic inhibitors of human neutrophil collagenase (MMP8). The presence of an aromatic residue both at the C2 malonic acid position and in the C-terminal tail for hydrophobic interactions with the surface-exposed S1 binding site and the S1' pocket of the enzyme, respectively, was found to be sufficient for submicromolar inhibition potencies. For optimal insertion of the aryl amide group into the hydrophobic S1' pocket, spacing of the C-terminal phenyl group by at least a 3C-chain was required. In view of these results the achiral indan-2, 2-dicarboxylic acid was used to mimic the 2-benzyl-2-methylmalonic acid residue, and its derivatization to the 3-phenylpropyl amide hydroxamate produced a potent, achiral, low-mass inhibitor of MMP8 (Ki = 0.3 microM), the binding mode of which was unambiguously determined by X-ray crystallographic analysis.

Binding Sites↗
PubMed1998

Met-195 of the cholecystokinin-A receptor interacts with the sulfated tyrosine of cholecystokinin and is crucial for receptor transition to high affinity state.

Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2. 8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10- and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation.

Animals↗
PubMed1998

Atomic mutations in annexin V--thermodynamic studies of isomorphous protein variants.

We have recently developed methods for specific and high-level replacement of methionine with 2-aminohexanoic acid, selenomethionine and telluromethionine as isosteric and atomic analogues for structural investigations of human recombinant annexin V. The variants formed isomorphic crystals and retained the parent three-dimensional fold and bioactivities. Folding parameters were determined from thermal and chemical unfolding to partially denatured states. Stabilities estimated from guanidinium chloride unfolding equilibria are not changed significantly for the atomic mutants (S-->Se-->Te) while the denaturation midpoint is shifted toward lower values with an increase of the m values at the increase of hydrophobicity. In contrast, stabilities in urea are considerably affected by the atomic substitutions, decreasing together with the m and [D]1/2 values. The methylene and selenium variants are identical within the limits of error of all measurements performed here. The physical parameters of the amino acid analogues and the values derived from the slopes of the unfolding data are highly correlated. This approach demonstrates how systematic variations in atomic number at the site of replacement (atomic mutations) can provide a method to probe specific folding properties of proteins.

Annexin A5↗
PubMed1998

Interactions of benzodiazepine derivatives with annexins.

Human annexins III and V, members of the annexin family of calcium- and membrane-binding proteins, were complexed within the crystals with BDA452, a new 1,4-benzodiazepine derivative by soaking and co-crystallization methods. The crystal structures of the complexes were analyzed by x-ray crystallography and refined to 2.3- and 3.0-A resolution. BDA452 binds to a cleft which is located close to the N-terminus opposite to the membrane binding side of the proteins. Biophysical studies of the interactions of various benzodiazepine derivatives with annexins were performed to analyze the binding of benzodiazepines to annexins and their effects on the annexin-induced calcium influx into phosphatidylserine/phosphatidylethanolamine liposomes. Different effects were observed with a variety of benzodiazepines and different annexins depending on both the ligand and the protein. Almost opposite effects on annexin function are elicited by BDA250 and diazepam, its 7-chloro-derivative. We conclude that benzodiazepines modulate the calcium influx activity of annexins allosterically by stabilizing or destabilizing the conducting state of peripherally bound annexins in agreement with suggestions by Kaneko (Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997) J. Mol. Biol., in press).

Amino Acid Sequence↗
PubMed1998

Design and synthesis of malonic acid-based inhibitors of human neutrophil collagenase (MMP8).

For most of the known synthetic inhibitors of matrix metalloproteinases (MMPs), a substrate-like binding mode was postulated on the basis of X-ray crystallographic structures of MMP/inhibitor complexes. Conversely, the malonic acid-based inhibitor (2R,S)-HONH-CO-CH(i-Bu)-CO-Ala-Gly-NH2 was found to bind in a surprisingly different manner. Using this compound as a new lead structure, the interaction sites with human neutrophil collagenase (MMP8) were optimized with a series of iteratively designed analogues and with the help of X-ray structural analysis of selected inhibitors to finally produce low molecular weight nonpeptidic compounds of 500-1000-fold improved inhibitory potency.

Crystallography, X-Ray↗