Investigation of conformational specificity at GPIIb/IIIa: evaluation of conformationally constrained RGD peptides.

RGD-containing proteins and peptides are known to bind to the platelet GPIIb/IIIa receptor and inhibit platelet aggregation. That a conformational component to the specificity exists is suggested by significantly lower activity of linear RGD analogs relative to closely related cyclic peptides and small proteins containing the RGD sequence. Recently, conformations for a suite of RGD containing cyclic peptides have been defined by NMR-based methods and, for one molecule, by X-ray diffraction. We report here the NMR-based conformational analysis of an additional cyclic peptide, cyclo(Pro-Arg-Gly-Asp-D-Pro-Gly), and compare the conformational variations in the suite of peptides and related analogs. Biological activity data for these peptides shows a preference of the platelet GPIIb/IIIa receptor for one conformation of the RGD sequence, but suggests its ability to bind a second, distinct conformation.

Structure-function analysis of human transforming growth factor-alpha by site-directed mutagenesis.

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

NMR study on solution structure of the site-specific mutant Leu48----Ala transforming growth factor alpha.

The NMR spectra of the Leu48----Ala mutant of human transforming growth factor alpha were compared to that of the wild-type. All chemical shift changes are less than or equal to 0.02 ppm with the exception of resonances associated with residues 47, 48 and 50 (all less than or equal to 0.07 ppm). Minimal changes were observed for NOEs associated with residues Val1 to His45. The weakening of some NOEs associated with the region Ala46-Ala50 may suggest a slightly increased flexibility for this region. Refinement of the previously calculated wild-type structures using distance constraints derived from the L48A mutant had little overall effect. Leu48-Ala50 is ill-defined for both wild-type and mutant proteins. These results suggest that Leu48 has no structural role and thus must be an important factor in the protein-receptor interface.

Solution structures of human transforming growth factor alpha derived from 1H NMR data.

The 600-MHz 1H NMR spectrum of the des-Val-Val mutant of human transforming growth factor alpha (TGF-alpha) was reassigned at pH = 6.3. The conformation space of des-Val-Val TGF-alpha was explored by distance geometry embedding followed by restrained molecular dynamics refinement using NOE distance constraints and some torsion angle constraints derived from J-couplings. Over 80 long-range NOE constraints were found by completely assigning all resolved cross-peaks in the NOESY spectra. Low NOE constraint violations were observed in structures obtained with the following three different refinement procedures: interactive annealing in DSPACE, AMBER 3.0 restrained molecular dynamics, and dynamic simulated annealing in XPLOR. The segment from Phe15 to Asp47 was found to be conformationally well-defined. Back-calculations of NOESY spectra were used to evaluate the quality of the structures. Our calculated structures resemble the ribbon diagram presentations that were recently reported by other groups. Several side-chain conformations appear to be well-defined as does the relative orientation of the C loop to the N-terminal half of the protein.

NMR studies of an oligonucleotide with an unusual structure induced by platinum anti-cancer drugs.

The 31P NMR spectra of Pt(en)[d(T1A2T3G4G5G6T7A8C9C10C11A12T13A14)] (14-mer) and Pt(en)[d(A2T3G4G5G6T7A8C9C10C11A12T13)] (12-mer) (en = ethylenediamine) each contain two signals far downfield (ca. -2.9 and -2.6 ppm from trimethylphosphate standard), two signals slightly downfield, and at least one signal slightly upfield of the normal range (ca -4.0 to -4.4 ppm). This pattern suggested a distorted structure. The unusual 31P signals of the 12-mer were assigned by analogy to signals of the 14-mer previously assigned by 17O-labeling methods. A combination of heteronuclear multiple-quantum coherence, one-dimensional and two-dimensional nuclear Overhauser effect (1D and 2D-NOE) and homonuclear shift correlation spectroscopy (COSY) experiments assigned all aromatic 1H signals of the 12-mer except H8 of G5 or G6. One of these H8 signals is missing from the spectrum and the nucleotide is labeled Gm. The other H8 is the most downfield signal and has a strong NOE to its H1'. Since this strong NOE indicates that this nucleoside exists in a syn conformation, it is labeled Gs. A strong NOE was observed between the Gs and A8 H8 signals. Several lines of evidence suggested a hairpin-like structure with a loop region (G6T7A8C9) and a stem region involving A2T3G4G5 and C10C11A12T13. The 31P signals for the stem region are within or slightly outside the normal range. 3JH3'-P values (3-6 Hz), measured by a 2D-J experiment, of stem nucleotides were characteristic for a DNA duplex. Imino signals for base pairs A2T13, T3A12, G4C11, and probably G5C10, and the observation of internucleotide NOE connectivities for these nucleotides (e.g. between an H8 signal and the H1' signal of the 5' nucleotide) suggested a right-handed helical structure. For the loop region, a distorted sugar-phosphate backbone is indicated by far downfield positions of the G5pG6 and A8pC9 31P signals, the 3JH3'-P values for C9p (8.0 Hz) and A8p (6.8 Hz), and the absence of H3'-P coupling for G5p. In the loop region, no imino signals or internucleotide NOEs characteristic of a right-handed duplex were observed. However, A8H8, C9H6, and C10H6 each exhibited unusual internucleotide NOEs to the H4' signal of the 5' residue. NOE crosspeaks between T7 1H signals and signals attributed to sugars of the Gs and Gm suggested that the T7 moiety is located within the space encircled by the loop. The few NOE crosspeaks, pH dependence, and Cu2+ broadening of C9 1H signals indicate an isolated location accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)