Structures of the Contryphan Family of Cyclic Peptides. Role of Electrostatic Interactions in cis−trans Isomerism^,

Abstract

The contryphan family of cyclic peptides, isolated recently from various species of cone shell, has the conserved sequence motif NH(3)(+)-X(1)COD-WX(5)PWC-NH(2), where X(1) is either Gly or absent, O is 4-trans-hydroxyproline, and X(5) is Glu, Asp, or Gln. The solution structures described herein of two new naturally occurring contryphan sequences, contryphan-Sm and des[Gly1]-contryphan-R, are similar to those of contryphan-R, the structure of which has been determined recently [Pallaghy et al. (1999) Biochemistry 38, 11553-11559]. The (1)H NMR chemical shifts of another naturally occurring peptide, contryphan-P, indicate that it also adopts a similar structure. All of these contryphans exist in solution as a mixture of two conformers due to cis-trans isomerization about the Cys2-Hyp3 peptide bond. The lower cis-trans ratio for contryphan-Sm enabled elucidation of the 3D structure of both its major and its minor forms, for which the patterns of (3)J(H)(alpha)(HN) coupling constants are very different. As with contryphan-R, the structure of the major form of contryphan-Sm (cis Cys2-Hyp3 peptide bond) contains an N-terminal chain reversal and a C-terminal type I beta-turn. The minor conformer (trans peptide bond) forms a hairpin structure with sheetlike hydrogen bonds and a type II beta-turn, with the D-Trp4 at the 'Gly position' of the turn. The ratio of conformers arising from cis-trans isomerism around the peptide bond preceding Hyp3 is sensitive to both the amino acid sequence and the solution conditions, varying from 2.7:1 to 17:1 across the five sequences. The sequence and structural determinants of the cis-trans isomerism have been elucidated by comparison of the cis-trans ratios for these peptides with those for contryphan-R and an N-acetylated derivative thereof. The cis-trans ratio is reduced for peptides in which either the charged N-terminal ammonium or the X(5) side-chain carboxylate is neutralized, implying that an electrostatic interaction between these groups stabilizes the cis conformer relative to the trans. These results on the structures and cis-trans equilibrium of different conformers suggest a paradigm of 'locally determined but globally selected' folding for cyclic peptides and constrained protein loops, where the series of stereochemical centers in the loop dictates the favorable conformations and the equilibrium is determined by a small number of side-chain interactions.

The Metal-free and Calcium-bound Structures of a γ-Carboxyglutamic Acid-containing Contryphan from Conus marmoreus, Glacontryphan-M

“…These results are similar to the random coil chemical shift deviations reported for contryphan-R at 5°C and pH 5.8 (7). These results suggest that in the absence of calcium, synthetic glacontryphan-M expresses the highly constrained, disulfidebridged, contryphan loop, which has been observed in previous NMR studies of contryphans-R, Sm, and Vn (5,7,10).…”

“…Contryphans characterized to date are a mixture of two conformers in solution due to cis-trans isomerization of the N-terminal Cys-Pro peptide bond (5). In contrast to previous structure/function studies of contryphan-Sm in which the cis-trans ratio of the Cys-Pro peptide bond was shown to be dependent on stabilizing electrostatic interactions between an N-terminal glycine residue and a glutamine that is positioned between D-Trp and Pro 9 (5), we observed no isomerization in glacontryphan-M in the absence or presence of calcium.…”

“…The nine contryphans characterized to date share the conserved sequence motif, CP*(D-W or D-L)XPWC, that includes a tryptophan or leucine in the D-conformation, a disulfide bond between the two cysteines, and in some cases hydroxylation of the proline preceding the D-Trp residue, Pro*. The N-terminal Cys-Pro peptide bond exhibits cis-trans isomerization in the contryphans characterized to date; however, the more abundant cis isomer is believed to be the functionally relevant conformer (5). The contryphan motif CP*(D-W or L)XPWC is a robust structural scaffold, that maintains the backbone structure and ␣-␤ bond vector orientation independent of the amino acid that is substituted at residue X (6).…”

The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

“…All spectra were acquired at 5°C to slow the peptide tumbling and to favour the lowest-energy conformations. 34,35 A discussion of the estimation of backbone structural characteristics from secondary chemical shifts and NOE patterns can be found in the Supplementary Material. NOE volumes were measured using Sparky 36 and referenced internally to fixed distances (pairs of H β s, Gly H α s, Trp ring neighbours) through the ISPA approximation, 37 with an added margin of 0.5 Å to account for uncertainties.…”

Post-translational Modifications of Arabinogalactan-peptides of Arabidopsis thaliana

“…For example, AGP21 has a unique N-terminal sequence ATVEA that was found in five fractions, A7 to A11 (Table I). This is likely because of cis-trans isomerization of Hyp that can lead to temperature-sensitive conformation changes that result in different retention times on RP-HPLC (40,41).…”