Twists or turns: stabilising alpha vs. beta turns in tetrapeptides

Abstract

Protein-protein interactions involve hotspots as small as 4 sequential amino acids. Corresponding tetrapeptides have no structure in water. Here we report linking side chains of amino acids X and Z to form 24 cyclic tetrapeptides, cyclo-[XAAZ]-NH₂, and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots. 2D NMR spectra allowed determination of 3D structures for 14 cyclic tetrapeptides in water. Five formed two (i, i + 3) hydrogen bonds and a beta/gamma (6, 7) or beta (9, 19, 20) turn; eight formed one (i, i + 4) hydrogen bond and twisted into a non-helical (13, 18, 21, 22, 24) or helical (5, 17, 23) alpha turn; one was less structured (15). A beta or gamma turn was favoured for Z = Dab, Orn or Glu due to a χ1 gauche (+) rotamer, while an alpha turn was favoured for Z = Dap (but not X = Dap) due to a gauche (-) rotamer. Surprisingly, an unstructured peptide ARLARLARL could be twisted into a helix when either a helical or non-helical alpha turn (5, 13, 17, 18, 21-24) with Z = Dap was attached to the N-terminus. These structural models provide insights into stability for different turns and twists corresponding to non-regular folds in protein hotspots.

Fig. 1. 7-, 10-, 13-membered hydrogen bonded rings define γ-, β- and α-turns respectively (left) that potentially might be stabilised by cyclisation via side chains (right).

Fig. 2. CD spectra of cyclic peptides (150 μM) in aqueous phosphate buffer (10 mM, pH 7.4, 25 °C). (A) 1–4. (B) 5–8. (C) 9–12. (D) 13–16. (E) 17–20. (F) 21–25. Standard 1-letter codes for l-amino acids unless otherwise indicated. Dap = l-2,3-diaminopropionic acid; Dab = l-2,4-diaminobutyric acid; O = l-ornithine; Succ = succinic acid; Glut = glutaric acid; IsoD and Isod are l- and d-aspartic acid respectively, where a side chain carbonyl instead forms the amide bond with the backbone nitrogen atom of the next residue.

Fig. 3. NMR summaries, 20 lowest energy structures, Ramachandran plots and turn types in water for fourteen cyclic tetrapeptides: 5–7, 9, 13, 15, 17–24 in H₂O/D₂O (9 : 1) at 298 K. Compounds 5, 13, 17, 18, 21–24 form one hydrogen bond; while 6, 7, 9, 15, 19, 20 form two hydrogen bonds.

Fig. 4. (A) Rotamer populations for residue Z4 from 50 ns MD simulations of cyclic peptides 5–20. NMR structures of: (B) 7 and 5 show how the χ1 angle of residue Z4 controls β(III)-turn vs. α-turn structures.

Fig. 5. Average backbone solution structures of 5–7, 9, 15, 17–21 and 23–24 (green; residues: A, A, X/Z) superimposed on the three central, consecutive, residues of an α-helix, α-turn, β-III turn and β-I turn from protein crystal structures (yellow). The linkers in these compounds are coloured grey.