Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding

Yu-Kuo Wang,Feng-Pai Chou,Chin-Yuan Chang,Sheng-Cih Huang,Bak-Sau Yip,Man-Jun Liao,Tung-Kung Wu,Wan-Ting Huang,Thomas Tien-Hsiung Li

doi:10.1038/s41598-019-46354-x

Abstract

Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell–cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh–TDH), a pore-forming toxin. The toxin crystalized in the same space group of P21212, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer–I, –II, and –III. A central pore with comparable depth of ~50 Å but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.

Highlights

Pore–forming toxins (PFTs), a class of virulence factors widely distributed in both prokaryotic and eukaryotic organisms, are common and effective biological weapons used to attack host cells through disruption of cellular membranes[1,2,3,4]
The Gh–TDH Oligomer–I structure was indistinguishable from the reconstituted Vp–TDH tetramer structure; whereas Oligomer–II and –III structures gave further insight into multiple Gh–TDH tetrameric oligomerization conformations in erythrocytes binding; the potential interfaces, in addition to those originally found in Oligomer–I structure, in Gh–TDH protomer for protein–protein interaction; and above all, the common TDH dimeric motif found within these Gh–TDH tetrameric pore–forming architectures as a minimum unit in cell membrane binding and probably hemolytic activity
Several lines of biophysical studies and biochemical data have demonstrated that Vp–TDH in solution is tetrameric with C4 symmetry, crystallographic analysis showed the asymmetric unit to contain a Vp–TDH protomer[22,23]

Summary

Introduction

Pore–forming toxins (PFTs), a class of virulence factors widely distributed in both prokaryotic and eukaryotic organisms, are common and effective biological weapons used to attack host cells through disruption of cellular membranes[1,2,3,4]. Fukui et al studied the “Arrhenius effect” of TDH, characterized by paradoxical responses to heat detoxification at ~60–70 °C and reactivation by additional heating above 80 °C23 This observation reveals the reversible fibrillar aggregation nature of TDH similar to amyloid fibrils formation, which implies the possible involvement of multiple interfaces of the TDH protomer in oligomerization[24]. The Gh–TDH Oligomer–I structure was indistinguishable from the reconstituted Vp–TDH tetramer structure; whereas Oligomer–II and –III structures gave further insight into multiple Gh–TDH tetrameric oligomerization conformations in erythrocytes binding; the potential interfaces, in addition to those originally found in Oligomer–I structure, in Gh–TDH protomer for protein–protein interaction; and above all, the common TDH dimeric motif found within these Gh–TDH tetrameric pore–forming architectures as a minimum unit in cell membrane binding and probably hemolytic activity

Methods

Results

Conclusion