Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.

Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.

Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae alpha-factor.

Magnaporthe grisea is a fungal pathogen with two mating types, MAT1-1 and MAT1-2, that forms a specialized cell necessary for pathogenesis, the appressorium. Saccharomyces cerevisiae alpha-factor pheromone blocked appressorium formation in a mating type-specific manner and protected plants from infection by MAT1-2 strains. Experiments with alpha-factor analogs suggest that the observed activity is due to a specific interaction of alpha-factor with an M. grisea receptor. Culture filtrates of a MAT1-1 strain contained an activity that inhibited appressorium formation of mating type MAT1-2 strains. These findings provide evidence that a pheromone response pathway exists in M. grisea that can be exploited for plant protection.

Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest.

Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.

Towards determination of the structure of the Saccharomyces cerevisiae a-factor: an acylated pentadecapeptide blocks a-factor activity.

Putative a-factor peptides YIIKGVFWADP, YIIKGVFWANP, YIIKGLFWADP, YIIKGLFWANP, YIIKGVFWDPA, and YIIKGVFWDPACVIA and several peptide derivatives were synthesized and were found to be inactive in growth arrest assays, yet they blocked the activity of biological a-factor. Antagonism was greatest with YIIKGVFWDPAC(palmitoyl)VIA. Thus, the structure of a-factor may be a lipopeptide resembling this palmitoylated pentadecapeptide.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium.

The transport of [14C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a Km of 5.3-10(-7)M and a V of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting Ki values between 10(-8) and 10(-7) M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.

Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles.

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator m-chlorocarbonylcyanide phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.

Mutations affecting ligand specificity of the G-protein-coupled receptor for the Saccharomyces cerevisiae tridecapeptide pheromone.

Random mutations were generated in the G-protein-coupled receptor (Ste2p) for the tridecapeptide pheromone (alpha-factor) of Saccharomyces cerevisiae. These mutants were screened for variants that responded to antagonists. Because multiple mutations were detected in each mutant receptor recovered from the screen, site-directed mutagenesis was used to create single-site mutant receptors. Three receptors containing mutations F55V, S219P, and S259P were analyzed for their biological responses to various alpha-factor analogs and for their ligand binding profiles. Cells expressing each of the mutant receptors responded to alpha-factor as well as or better than wild-type cells in a growth arrest assay. In contrast, the binding of alpha-factor to the F55V and S219P mutant receptors was at least 10-fold reduced in comparison to wild-type receptor indicating a complex non-linear correlation between binding affinity and biological activity. Cells expressing mutant receptors responded to some normally inactive analogs in biological assays, despite the fact that these analogs had a low affinity for Ste2p. The analysis of these mutant receptors confirms previous findings that the first and sixth transmembrane regions of Ste2p are important for ligand interaction, ligand specificity, and/or receptor activation to initiate the signal transduction pathway. Changes in binding affinity of pheromone analogs to wild-type and mutant receptors indicate that residue 55 of Ste2p is involved with both ligand binding and signal transduction.

Lipid-mediated a-factor interactions with artificial membranes.

Our understanding of the cellular export of a-factor and its interaction with the receptor do not yet allow for a description of the phenomena on a molecular level. Synthesis of a-factor analogs and biophysical studies of the lipopeptides in the presence of artificial membranes provide insights which can be analyzed with respect to the biological potency of the molecules. It is through the study of the interaction of the lipopeptides with membranes at varying levels of complexity that we will be able to develop a molecular description of the biological processes.

Synthesis and biological evaluation of dipeptidyl and tripeptidyl polyoxin and nikkomycin analogues as anticandidal prodrugs.

Nine analogues (1-5, 9-12) of the peptidyl nucleoside antibiotics nikkomycin and polyoxin were synthesized and tested for their biological properties against different strains of the pathogenic yeast Candida albicans. The tripeptidyl series of analogues (1-5) was designed to behave as prodrugs, releasing a toxic moiety upon enzymatic hydrolysis inside the cell. The dipeptidyl series (9-12) was designed as double-targeted drugs, being themselves toxic and releasing a toxic amino acid upon hydrolysis. All the analogues were prepared by coupling suitably protected amino acid p-nitrophenyl esters to 1-(5'-amino-5'-deoxy-alpha-D-allofuranuronosyl)uracil (UPOC) or the corresponding polyoxins and nikkomycins, with subsequent removal of the protecting group. Improved coupling yields were observed when DMSO was used as the solvent. Products were purified with use of reversed-phase HPLC and, in one case, diastereomeric products (compound 11) were resolved by using this procedure. One of the tripeptidyl nikkomycins behaved as a prodrug but none of the compounds, as measured by in vitro testing, proved more effective than nikkomycin as an anticandidal agent.

Anticandidal activity of 5-fluorocytosine-peptide conjugates.

An approach to the development of new anticandidal drugs is described that employs peptides as carriers of toxic agents into cells. 5-Flurorcytosine (5-FC) was chosen as a toxic agent with which to prepare 5-FC-peptide conjugates as models to test the carrier proposal. Model compounds were synthesized and then tested for antiyeast activity against S. Cerevisiae 9763, C. albicans 1-V, C. albicans WD 18-4, and C. Krusei 1-T. The 5-FC derivatives showed antiyeast activity comparable to 5-FC in all strains except C. krusei 1-T, in which case the compounds were less active. The solution stabilities of 5-FC conjugates at 37 degrees C were tested in the same growth medium used for susceptibility testing. The results indicated a range of stabilities where the half-life (t1/2) = 0.3--17.6 h. These results and those obtained in the susceptibility testing suggest extracellular hydrolysis and indicate that the type of linkage used to conjugate 5-FC to peptides will not provide appropriate compounds to evaluate the peptide-carrier concept.

Synthesis and biological properties of chitin synthetase inhibitors resistant to cellular peptidases.

The synthesis and biological properties of seven polyoxins (4-10) designed to avoid peptidase hydrolysis in Candida albicans are presented. Five dipeptidyl and two tripeptidyl polyoxin analogues were synthesized by coupling an amino acid active ester or azlactone to uracil polyoxin C (2) or polyoxin D (1), subsequent removal of the protecting group, and purification by preparative HPLC. A new and novel route for introducing an n-propyl group onto the alpha-amino group of peptides is reported. With the exception of a carboxamide derivative, 8, all analogues were resistant to hydrolysis by a cell extract or permeabilized cells of Candida. Chitin synthetase inhibition constants were determined for 4-10 and the KI values ranged from 7.15 X 10(-6) M for octanoyl-phenylalanyl-polyoxin D (10) to 1.06 X 10(-3) M for D-tryptophanyl-uracil polyoxin C (6). These novel polyoxins do not compete with the transport of either peptides or uridine into the cell. Millimolar concentrations of compounds 4-10 are required to inhibit growth, cause morphological alterations, or reduce the viability of C. albicans.

Synthesis and anticandidal properties of polyoxin L analogues containing alpha-amino fatty acids.

Analogues of polyoxin L containing amino acids with saturated fatty acid like side chains were synthesized from the benzyloxycarbonyl-protected alpha-amino fatty acid p-nitrophenyl ester and uracil polyoxin C. Transfer hydrogenolysis using palladium black and formic acid gave diastereomeric, dipeptidyl polyoxin L analogues containing alpha-aminooctanoic acid (3), alpha-aminododecanoic acid (4), or alpha-aminohexadecanoic acid (5) as the amine terminal residue in 40-60% yield. Diastereomers of 3 and 5 were resolved by using high-performance liquid chromatography on a reversed-phase column and designated as 3a, 3b and 5a, 5b. Analogues 3-5 were excellent inhibitors of chitin synthetase from Candida albicans; 4, the best inhibitor, had an ID50 of 0.5 microM. The L,L diastereomers of 3 and 5 were 1-2 orders of magnitude more potent chitin synthetase inhibitors than their D,L homologues. None of the synthetic polyoxin L analogues inhibited transport of trimethionine, but 3a, 4, and 5b caused decreases of 71%, 87%, and 83%, respectively, in the initial rate of uptake of dileucine. Compounds 3-5 were significantly more stable to peptidase degradation than polyoxin L analogues containing naturally occurring alpha-amino acids. Compound 4 inhibited growth of C. albicans in culture at 40-80 micrograms/mL. All other analogues were less potent antifungals. The results suggest that synthetic polyoxins can be designed to have increased affinity for a peptide transport system and to have increased stability against intracellular degradation in C. albicans.

Conformation-activity relationship of sweet molecules. Comparison of aspartame and naphthimidazolesulfonic acids.

The shape of the active site of the receptor for sweet molecules was previously defined on the basis of a combination of both rigid (saccharins) and flexible (aspartame) molds. In this paper, the sweetness receptor is refined with use of the shapes of 3-anilino-2-styryl-3H-naphtho[1,2-d]imidazolesulfonate (sweet) and of 3-anilino-2-phenyl-3H-naphtho[1,2-d]imidazolesulfonate (tasteless), two large and almost completely rigid tastants. The minimum-energy conformations of the flexible portions of these tastants have been determined by using a detailed conformational analysis based on ab initio calculations. The refined receptor site is still consistent with all previously examined sweet molecules. In order to unequivocally assign the prochiral beta-CH2 protons of the Phe moiety of aspartame, (2S,3S)-[2H]-alpha-L-Asp-L-PheOMe was synthesized and examined by 500-MHz 1H NMR spectroscopy. The results indicate that the minimum-energy conformation for aspartame in water, DMSO-d6, and CDCl3 (as a crown ether complex) is different from that originally proposed (FIIDII instead of FIDII, according to a notation referred to the side chains). Although this conformation is not directly consistent with the shape of the sweet receptor, the interconversion of FIIDII to FIDII was found to require only 1 kcal/mol. Furthermore, a 120-ps molecular dynamics simulation in vacuo confirms the high flexibility of aspartame and the accessibility of the FIDII conformer whose topology is fully consistent with our model.

Anticandidal activity of pyrimidine-peptide conjugates.

The ability of conjugates of peptides and 5-fluorocytosine or 5-fluoroorotic acid to enter Candida albicans was investigated. A number of conjugates of 5-fluoroorotic acid and peptides were synthesized using 1-(ethoxy-carbonyl)-2-ethoxy-1,2-dihydroquinoline as the coupling agent. Orotyl-L-leucyl-L-leucine, 5-fluoro-4-(N-succinamoyl-L-alanyl-L-leucine)-2(1H)-pyrimidinone [a 5-fluorocytosine derivative], and 5-fluoroorotyl-L-leucyl-L-leucine all inhibited the uptake of trimethionine into C. albicans WD 18-4. Inhibition by 5-fluoroorotyl-L-leucyl-L-leucine was competitive as judged using double-reciprocal plots. Evaluation of minimum inhibitory concentrations of peptide-5-fluorocytosine conjugates suggest that these conjugates enter C. albicans in the intact form. These results provide the first experimental evidence that peptides can carry pyrimidines into a eukaryote.

Design of anticandidal agents: synthesis and biological properties of analogues of polyoxin L.

Six analogues of polyoxin L were synthesized from uridine. All of these analogues inhibited chitin synthetase from Candida albicans. Derivatization of the amine terminus of the polyoxin analogues resulted in loss of activity, and analogues containing aromatic amino acid residues were the most efficient inhibitors of chitin synthetase. The concentration of tryptophanyl uracil polyoxin C, 8, which caused 50% inhibition of chitin synthetase activity, was 1.6 X 10(-6) M. This was virtually identical with the activity found for polyoxin D. None of the inhibitors effectively competed with the entry of (Met)3 into C. albicans. All of the analogues caused severe morphological distortions of the yeast in culture, and a number of analogues killed C. albicans at millimolar concentrations. The results suggest that chitin synthetase inhibitors may have potential as anticandidal drugs.

Analysis of ligand-receptor cross-linked fragments by mass spectrometry.

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.