D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function.

Christoph Sonderegger,Laura Burtscher,Nick D Read,Gyula Batta,Alberto Muñoz,Florentine Marx,Zoltán Gáspári,Dorottya Hajdu,Ádám Fizil,Eugene A Permyakov

doi:10.1371/journal.pone.0169920

Abstract

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

Highlights

Antimicrobial proteins (AMPs) are gaining increased attention as promising new therapeutics to prevent and/or treat microbial infections
After three rounds of single spore isolation, positive transformed P. chrysogenum PAFD19S clones were tested for best protein production over a time course of 96 hours before one positive clone was selected for the highest secretion of PAFD19S into the supernatant
Our study sheds important new light on the structural and mechanistic basis of the antifungal protein PAF, which may support the understanding of the function of other related antifungal proteins

Summary

Introduction

Antimicrobial proteins (AMPs) are gaining increased attention as promising new therapeutics to prevent and/or treat microbial infections. One of the best-studied bio-molecules in structure and function is the antifungal protein PAF from the β-lactam producer Penicillium chrysogenum [4]. It is a prepro-protein which is processed before secretion and the mature PAF consists of 55 amino acids (Fig 1A) [4]. The β-strands are connected by four solvent exposed loops which show increased mobility and structural heterogeneity (Fig 1B) [7,8,9] These features point towards an important role of the loop regions in possible protein-host interactions and PAF toxicity [8]. We found in the PAF loop regions 2 and 3 a recurring asparagine-aspartate or aspartate-asparagine sequence preceding or following a lysine residue (Asn18-Asp in loop 2, Asp32-Asn and Asp39-Asn in loop 3) [7]

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