Solution Structure Determination of Four Diploptera punctata Allatostatins by NMR Spectroscopy and Molecular Modeling

Abstract

In 1989 a peptide showing an inhibitory effect against biosynthesis of juvenile hormone III (allatostatic effect) was found in cockroach Diploptera punctata. Its sequence was determined and named allatostatin. It has an amidated Cterminal. Four years later its precursor polypeptide was found, which was composed of 370 amino acids (NCBI access number P12764). Since the first discovery of allatostatin, three more analogues were known and named allatostatin I, II, III, and IV. They have a common Cterminal sequence, Tyr-Xaa-Phe-Gly-Leu-NH2. Based on the fact that there are thirteen common sequences in allatostatin precursor polypeptide, allatostatins of the same number were expected to be found. The precursor peptide isolated from cockroach D. punctata and the primary sequences of thirteen allatostatins are shown in Figure 1. They were named according to the order of positions placed in their precursor. Allatostatin I, II, III, and IV were renamed 7, 9, 8, and 5, respectively. The 50% effective doses against biosynthesis of juvenile hormone III of Diploptera punctata allatostatin 7 (DpAST7), Dp-AST9, Dp-AST8, and Dp-AST5 found in the early study are 4.1 × 10, 7.2 × 10, 9.4 × 10, and 1.6 × 10 M, respectively. The numbers of residues of DpAST7, Dp-AST9, Dp-AST8, and Dp-AST5 are 13, 10, 9, and 8, respectively. Therefore, there is no relationship between the number of residues and the effective doses. To provide basic information for the allatostatic effect, authors carried out the structural study of four D. punctata allatostatins using Nuclear Magnetic Resonance (NMR) spectroscopy and molecular modeling. The NMR data of Dp-AST7 obtained in both aqueous solution (90%H2O/10%D2O) and TFE/water binary solution (50%trifluoroethanol-d3/50%H2O) were assigned based on the standard procedures. Even though all of the backbone amide cross peaks were not observed in COSY and TOCSY, the chemical shifts of the residues could be assigned from the interpretation of the cross peaks between amide protons and alpha protons. In order to confirm the assignments, the sequential walk of the same region was carried out in the NOESY spectrum in TFE/water binary solution (Fig. 2). Assignments of NMR data of Dp-AST7 in both aqueous solution and TFE/water solution are listed in Table 1. While Dp-AST7 in aqueous solution did not show meaningful nOe