NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism

Abstract

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 degrees C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01, 0.89, and 1.01 A for backbone atoms and 1.82, 1.74, and 1.81 A for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel beta strands connected in a -1, -1, +3, +1 topology and highly twisted, defining a small and compact beta barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids which would be the basis of its biological function. On the other hand, a second, minor form of AFP has been detected. NMR data, together with results from mass spectrometry, chemical analysis, and sedimentation equilibrium, suggest that this species differs from the major form in the pairs of cysteines involved in the four disulfide bridges.

Survival Strategies of Yeast and Filamentous Fungi against the Antifungal Protein AFP

“…10 5 yeast cells were cultivated at 28°C in 150 l of YPD medium for 12-16 h until they reached the midlogarithmic growth phase. 1 M SYTOX Green and AFP (up to final concentrations of 400 g/ml) were added.…”

“…AFP is a 5.8-kDa small, cysteine-rich, amphipathic protein with a positive net charge and is secreted by A. giganteus especially under non-favorable growth conditions (9,10). The protein is active against filamentous fungi, including serious human and plant pathogens, but inactive against bacteria, yeast, plants, or mammalian cells and successfully protects plants from colonization or invasion of filamentous fungi (11,12).…”

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

“…This band is directly related to the native protein conformation but hampers calculations aimed to estimate secondary structure contents. The existence of different sets of disulfide bridges patterns within the same protein preparation further complicates this interpretation [19,21].…”

“…AFP has been thoroughly characterized from structural and spectroscopic points of view, including the resolution of its three-dimensional structure in solution [11,19]. It displays the characteristic features of an oligonucleotide/oligosaccharide binding (OB-fold) structural motif [20] being a small and compact β-barrel composed of five highly twisted antiparallel β-strands.…”

“…It displays the characteristic features of an oligonucleotide/oligosaccharide binding (OB-fold) structural motif [20] being a small and compact β-barrel composed of five highly twisted antiparallel β-strands. It should be also mentioned that cysteine pairing isomerism exists within its four disulfide bridges [19]. AFP is secreted as an inactive precursor (lf-AFP) containing an amino-terminal extension of six amino acids [12].…”