Photon Factory Activity Report 2018 #36 (2019)

BL-5A / 2014G190

Structure of the hemolytic toxin FraE helps to clarify the evolution of actinoporins.
1Jose M.M. Caaveiro1,2,*, Koldo Morante1, and Kouhei TSUMOTO1,3,*
Dept. of Bioengineering, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
2
Global Healthcare, Graduate School of Pharmacy, Kyushu University, Fukuoka 812-8582, Japan
3
Institute of Medical Science, The University of Tokyo, Tokyo 108-8639

We have expanded the structural catalogue of actinoporins, a family of highly potent hemolytic proteins belonging to theclass of pore-forming proteins. Herein we report the crystal structure of a new actinoporin that we termed FraE at 2.2 Åresolution. This structure improves our understanding of the evolution of this family of toxins.
1 Introduction
Actinoporins are pore-forming proteins produced by sea
anemones known for their widespread polymorphism [1].
Actinoporins have conserved functional sites to ensure thepreservation of their mechanism of pore-formation, acomplex process involving several different steps [2].
Among them, the oligomerization of ~8 units is driven byprotein-protein interactions that are conducive to theassembly of the functional pore in target membranes [3].
Previous studies identified variability in residues at theprotein-protein interaction surface. Herein we haveisolated a new variant of actinoporins termed FraE. By
using X-ray crystallography, we aim to understand if theprotein-protein surface and other key regions areconserved in the family of actinoporins.
2 Experimental
FraE was cloned into the pBAT4 vector. Expression of
FraE was carried out in Escherichia coli BL21(DE3).
Expression was induced with 0.5 mM IPTG for 20 hours
at 20 °C. Cells were harvested, lysed with a probesonicator, and the supernatant subjected to centrifugation.
The protein was subsequently purified by ion-exchange
and size-exclusion chromatography.
Crystals of FraE were obtained in a solution composedof 0.2 M sodium formate, 20% w/v PEG 3350 at pH 7.2.
Crystals were transferred to a solution of mother liquor
supplemented with 20 % (v/v) glycerol and fast-frozen inliquid nitrogen. Diffraction data was collected at BL-5A ofthe Photon Factory (Tsukuba, Japan) under cryogenic
conditions (100 K). The crystal structure of FraE wasdetermined by the method of molecular replacement using
the homolog protein FraC (PDB code 3VWI). The crystal
was determined at 2.2 Å resolution and deposited in theProtein Data Bank under entry code 6K2G.
3 Results and Discussion
A new actinoporin termed FraE was identified andcloned. We The crystal structure of FraE was determinedat a resolution of 2.2 Å. FraE exhibited similar structureand hemolytic properties to FraC and Eqt-II, two wellstudied actinoporins (Fig. 1) [2,4]. Based on these crystalstructures and their primary sequences, we identified
regions of high structural similarity such as the lipid
binding site and the protein-protein interaction surface, andother regions of significantly less similarity (Fig. 2).

Fig. 1: Structure of three actinoporins. Root-mean squaredeviation of their coordinates was less than 0.5 Å.

Fig. 2: Evolutionary variability of actinoporins.

We conclude that actinoporins are a homogeneous groupof proteins with very similar architecture, but also withlocal differences that may explain their target selectivity.
Acknowledgement
We are grateful to Dr. Juan Manuel González Mañas andDr. Augusto Bellomio for general advice. We thank thestaff of the Photon Factory for excellent technical support.
References[1] G. Anderluh et al., Acta Chim. Slov 58, 724 (2011).
[2] K. Tanaka et al., Nat. Commun. 6, 6337 (2015).
[3] K. Morante et al., FEBS Lett. 589, 1840 (2015).
[4] J.M.M. Caaveiro et al., Biophys J. 80, 1343 (2001).
*Corresponding
authors:
jose@phar.kyushu-u.ac.jp(JMMC), and tsumoto@bioeng.t.u-tokyo.ac.jp (KT)