Solution structure of PAFP-S: A new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide e xtracted from the seeds of Phytolacca americana, was determined using H-1 N MR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral rest raints derived from NOEs and coupling constants. The peptide has six cystei nes involved in three disulfide bonds. The previously unassigned parings ha ve now been determined from NMR data. The solution structure of PAFP-S is p resented as a set of 20 structures using ab initio dynamic simulated anneal ing, with an average RMS deviation of 1.68 Angstrom for the backbone heavy atoms and 2.19 Angstrom for all heavy atoms, respectively. For the well-def ined triple-stranded beta -sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 Angstrom. The global fold invo lves a cystine-knotted three-stranded antiparallel beta -sheet (residues 8- 10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta -reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of a n antifungal peptide that adopts a knottin-type structure. PAFP-S has an ex tended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32 , and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38 , and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological funct ion.