Synthesis, biological activity, and conformational analysis of peptidomimetic analogues of the Saccharomyces cerevisiae alpha-factor tridecapeptide.
Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor. To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted alpha-factor analogue WHWLQLK[(R)-gamma-lactam]QP[Nle]Y was more active than its lactam-containing diastereomeric homologue WHWLQLK[(S)-gamma-lactam]QP[Nle]Y and about equally active with the [Nle12]-alpha-factor in growth arrest and FUS1-lacZ gene induction assays. Both lactam analogues competed with tritiated [Nle12]-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone. The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones.
Zhang
Y L
YL
Department of Chemistry, The College of Staten Island, The Graduate School of The City University of New York 10314, USA.
Marepalli
H R
HR
Lu
H F
HF
Becker
J M
JM
Naider
F
F
D000577:Amides; D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D006131:Growth Inhibitors / Q000378:metabolism; D007769:Lactams / Q000378:metabolism; D000072235:Mating Factor; D018716:Molecular Mimicry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D010675:Pheromones / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000254:growth & development; D012996:Solutions; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors
D000577:Amides; D000596:Amino Acids; D006131:Growth Inhibitors; D007769:Lactams; D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D012996:Solutions; D014157:Transcription Factors; D000072235:Mating Factor