title: Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase.
abstract: The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.
authors.affiliation: Laboratory of Medicinal Chemistry, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Barchi
authors.forename: M
authors.initials: M
authors.lastname: Nomizu
authors.forename: A
authors.initials: A
authors.lastname: Otaka
authors.forename: P P
authors.initials: PP
authors.lastname: Roller
authors.forename: T R
authors.initials: TR
authors.lastname: Burke
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010456:Peptides, Cyclic / Q000737:chemistry*; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D011487:Protein Conformation; D013816:Thermodynamics; D018909:src Homology Domains
chemical_list: D008024:Ligands; D009842:Oligopeptides; D010456:Peptides, Cyclic; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)