Photon Factory Activity Report 2016 #34 (2017)

AR-NW12, 2014G190

Mechanism of recognition of a membrane-embedded epitopeby the anti-HIV broadly-neutralizing 10E8 antibody.
Jose M.M. Caaveiro,1,2,* Edurne Rujas,1,3 Jose Luis Nieva,3 and Kouhei Tsumoto1,4,*
1Dept. 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
Faculty of Science, UPV/EHU, and Biofisika, CSIC-UPV/EHU, P.O. Box 644, Bilbao 48080, Spain
4Institute of Medical Science, University of Tokyo, Tokyo 108-8639
The mechanism by which the MPER epitope of gp41 of HIV-1 is recognized by the broadly neutralizing antibody 10E8
at membrane interfaces is not known. We determined the crystal structure of 10E8 Fab complex in complex with thetransmembrane antigen in detergents. Our research clarifies the molecular mechanisms underlying broad neutralization
by 10E8, and guiding future vaccination strategies targeting the MPER antigen of HIV-1 [1].
1 Introduction
Despite of the innumerable advances in virology,
pharmacology and clinical fields throughout these years,>30 million people remain infected by HIV according tothe WHO. The high mutation rate of HIV-1 and its viral
cycle make this virus very difficult to eradicate once it
has infiltrated in the human body. The current
antiretroviral therapy has turned HIV infection into achronic disease, but it is unable to completely eradicate
the virus.
A promising therapeutic strategy consists in eliciting
vaccination responses in which broadly neutralizing
antibodies (bNAbs) are generated, although the efficiencyof the level of the responses are still unsatisfactory. 10E8
is a potent bNAb very efficient against nearly all known
strains of HIV-1 [2]. The advantageous properties of 10E8
could be used to devise novel strategies of anti-HIV-1
vaccination. However, there are fundamental properties of
10E8 that need to be addressed before it can be usedefficiently for that purpose. In this study, we determined
the influence of the cellular membrane in the mechanismof recognition using lipidic model systems and X-ray
crystallography.
2 Experiment
Crystals of the complex between 10E8 Fab (3 mg L−1)
and the peptide 10E8ep at a molar ratio of ~1:1.5(Fab:peptide) in the presence of 2.5 mM detergent
dodecylphosphocholine were grown in a solution of 50%(v/v) 2-methyl-2,4-pentanediol, 200 mM phosphate, and100 mM TRIS (pH 8.5) at 20 °C. A single protein crystal
was harvested and stored in liquid nitrogen. Datacollection was carried out at beamline AR-NW12 of thePhoton Factory in Tsukuba, Japan.
Isothermal titration calorimetry (ITC) was performed
with a VP-ITC instrument at 25 °C. Proteins weredialyzed overnight at 4 °C. Fab 10E8 (3 µM) was titrated
with peptide (40 µM). For differential scanningcalorimetry (DSC) the heat capacity was measured using
a VP-DSC scanning microcalorimeter. Samples of 10E8(10 µM) with and without peptide (15 µM) were heatedfrom 30 to 90 °C at a rate of 1 °C min−1.

3 Results and Discussion
We have determined the structure of 10E8 Fab incomplex with the membrane-proximal external region
peptide antigen (MPER) and significant portion of thetransmembrane domain (TMD) of gp41 (Fig. 1). The
dissection of the structural and the energetic factors
controlling the recognition of the epitope by the Fab inmodel membrane systems explains the potent
neutralization capabilities of the full-length antibody. We
propose that the helical scaffold of the MPER / TMD
region of gp41, strengthened by nonpolar interactions
with membrane lipids, is critical for the generation ofpotent anti-MPER broadly neutralizing antibodies [1].

Fig. 1: X-ray structure of the bNAb 10E8 withMPER/TMD peptide in micelles. The position of themembrane with respect to the complex is approximatedwith the lines at the bottom of the figure.
Acknowledgement
We acknowledge financial support from JSPS
KAKENHI-A 25249115 and KAKENHI-C 15K06962.
References[1] E. Rujas et al., Sci Rep. 6:38177 (2016).
[2] J. Huang et al. Nature 491, 406 (2012).
*jose@phar.kyushu-u.ac.jp, tsumoto@k.u-tokyo.ac.jp