Photon Factory Activity Report 2016 #34 (2017)

AR-NW12A/2014G190

Dual recognition of lipids and saccharides employing
a single binding site in a pore forming toxin
Jose M.M. Caaveiro,1,2,* Koji Tanaka,3 and Kouhei Tsumoto1,3,4,*
1
Dept. of Bioengineering, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan
2
Global Healthcare, Graduate School of Pharmacy, Kyushu University, Fukuoka 812-8582, Japan
3
Dept. of Chem. & Biotechnol., School of Engineering, University of Tokyo, Tokyo 113-8656, Japan
4
Institute of Medical Science, University of Tokyo, Tokyo 108-8639
Fragaceatoxin C (FraC) is a pore forming toxin (PFT) that interacts with biological membranes using a lipid binding
motif. Since FraC also interact with the carbohydrates, we investigated the basis of glycan recognition. Surprisingly, we
found that FraC engages glycans using the same, high-affinity, lipid-binding module. In conclusion, FraC has developed
dual recognition capabilities with a single binding motif.
1 Introduction
PFTs comprise a heterogeneous family of toxic
proteins that target foreign cellular membranes withoffensive (or defensive) purposes. For example, humanperforin is a protein of defensive purposes of the immune
system, whereas bacterial PFTs are potent virulence
factors causing cell death.
FraC is a small but very potent hemolytic PFT of ~20
kDa secreted by sea anemones, lethal to small marineorganisms. Membrane selectivity is achieved by its
affinity for the lipid sphingomyelin (SM) of themembrane, which acts as a binding receptor [1].
Interestingly, FraC experience a significant delay in sizeexclusion chromatography (SEC) due to a supposedlyinteraction with the carbohydrate matrix of the resin [2].
However, the putative binding site for carbohydrates ortheir effect on the function and structure of FraC are still
unknown.
2 Experiment
A solution of microcrystals was prepared like that
described for unbound FraC [1]. Large rod-shaped single
crystals of the complex of FraC and GlcNAc(6S) wereobtained after mixing 1 µl of protein at 9 mg ml-1 in 10
mM Tris–HCl and 50 mM GlcNAc(6S) (pH 7.4), 0.2 ml
solution of micro-seeds, and 1 µl of crystallization
solution composed of 19% PEG 3350, 200 mM
ammonium chloride and 100 mM Bis–Tris (pH 6.3).
Suitable crystals appeared after one month at 20°C.
Crystals were subsequently frozen in the presence of 20%
glycerol by immersion in liquid nitrogen. Data collection
was carried out at beamline AR-NW12A of the Photon
Factory (Tsukuba, Japan).
3 Results and Discussion
In this study we hypothesized that the single-domain
hemolysin FraC could recognize lipids and carbohydrates
by a mechanism different from that of more complex
multi-domain PFTs. We thus investigated the ability ofFraC to interact with carbohydrates from various pointsof view. We first employed carbohydrate arrays toidentify a population of saccharides containing a negative

charge for which FraC showed preferential binding. We
thus characterized the binding site of the sulfatedmonosaccharide GlcNAc(6S) by X-ray crystallography athigh resolution (1.5 Å). Surprisingly, the carbohydrate
pocket overlapped with the lipid-binding module of FraC
(Fig. 1). The binding was characterized by low affinity
and no remarkable specificity, suggesting that there could
be another, more specific glycan from marine animals,not included in the human glycan array used for thescreening step. In summary, we reveal a novel recognition
mechanism by an ancient PFT that could enhance theactivity of FraC in the natural marine environment [3].

Fig. 1: Crystal structure of FraC in complex with themonosaccharide GlcNAc(6S). (a) Sigma-A weighted 2fofc electron density map contoured at 1.5 s. (b)Comparison of the relative position of the lipid (green)and saccharide (magenta) in the bound conformation.
Acknowledgement
We acknowledge financial support from JSPS
KAKENHI-A 25249115 and KAKENHI-C 15K06962.
References[1] K. Tanaka et al., Nat. Commun. 6, 6337 (2015).
[2] A. Bellomio et al., Toxicon 869, (2009).
[3] K. Tanaka et al., Philos. Trans. R. Soc. Lond. B Biol.
Sci. 372: 20160216.
* jose@phar.kyushu-u.ac.jp, tsumoto@k.u-tokyo.ac.jp