A synthetic hexapetide designed to resemble a proteinaceous P-loop nest is shown to bind inorganic phosphate

The hexapeptide Ser-Gly-Ala-Gly-Lys-Thr has been synthesized and characterized. It was designed as a minimal soluble peptide that would be likely to have the phosphate-binding properties observed in the P-loops of proteins that bind the beta-phosphate of GTP or ATP. The beta-phosphate in such proteins is bound by a combination of the side chain e-amino group of the lysine residue plus the concavity formed by successive main chain peptide NH groups called a nest, which is favored by the glycines. The hexapeptide is shown to bind HPO42- strongly at neutral pH. The affinities of the various ionized species of phosphate and hexapeptide are analyzed, showing that they increase with pH. It is likely the main chain NH groups of the hexapeptide bind phosphate in much the same way as the corresponding P-loop atoms bind the phosphate ligand in proteins. Most proteinaceous P-loops are situated at the N-termini of alpha-helices and this observation has frequently been considered a key aspect of these binding sites. Since the hexapeptide in isolation is unlikely to form even a short alpha-helix, this suggests that being at the N-terminus of an alpha-helix is not essential for phosphate binding. An unexpected finding about the hexapeptide-HPO42- complex is that the side chain e-amino group of the lysine occurs in its deprotonated form, which appears to bind HPO42- via an N•••H-O-P hydrogen bond .