Photon Factory Activity Report 2017 #35 (2018)

BL-5A, AR-NE3A/2013G738, 2014G190

Intramolecular H-bonds facilitate the recognition of a flexible peptide by an antibody.
1Jose M.M. CAAVEIRO,1,2,* Kazuhiro MIYANABE,3 and Kouhei TSUMOTO1,3,4,*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
Dept. of Chem. & Biotechnol., School of Eng., The University of Tokyo, Tokyo 113-8656, Japan
4
Institute of Medical Science, The University of Tokyo, Tokyo 108-8639We have shown that intramolecular H-bonds transiently stabilize the conformation of a flexible peptide in solution. The
stabilized conformation of the peptide in solution is consistent with the conformation of the peptide bound to its specificantibody as shown here by X-ray crystallography. Ablation of these intramolecular H-bonds modulates the affinity of thepeptide for the antibody by up to 2 kcal mol-1 of free energy. These results reveal an important role of intramolecular Hbonds in antibody binding and have implications for designing efficient vaccines from short peptides.
1 IntroductionShort peptides are employed to generate antibodies byimmunization procedures, such as in vaccines. Since short
peptides do not possess stable secondary structure insolution, it is unclear how antibodies mature andrecognize such flexible antigens. In particular, we areinterested to understand the role of intramolecular Hbonds in antibody recognition, and in particular their
possible role in the transient stabilization ofconformations of the peptide in the unbound state (insolution) and their thermodynamic consequences.
X-ray crystallography, MD simulations andcalorimetric techniques, all high resolution techniques,
were employed to address this question. In this report wedescribe the preparation and determination of highresolution crystal structures of the peptide pep1 (andmutations) derived from the therapeutically relevant
human protein CCR5 (co-receptor of HIV-1) with its
specific antibody 4B08.
2 Experiment
Recombinant scFv antibody 4B08 and mutants wereobtained from inclusion bodies produced in Escherichia
coli [1]. The refolded protein was purified by Ni2+-affinity
chromatography and by size exclusion chromatography.
Antibody 4B08 (100 µM) dialyzed in MES buffer (10mM MES, 30 mM NaCl, pH 5.9) was mixed with peptide(200 µM). Crystals of the complex were obtained in 1.8–
2.4 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.9–6.5 at 20 °C by
the hanging-drop vapor diffusion method. The crystals
were briefly transferred to mother liquor solution
supplemented with 30% glycerol prior to their storage inliquid N2 until data collection.
Diffraction data were collected at beamlines BL5A andAR-NE3A of the Photon Factory (Tsukuba, Japan) undercryogenic conditions (100 K). The resolution achieved forwild type, mutant N3A and T6A was 1.35, 1.96 and 1.35
Å, respectively. The coordinates and structure factors
have been deposited in the protein data bank (PDB) under
entry numbers 5YD3 (wild type), 5YD5 (N3A), and5YD4 (T6A).

3 Results and Discussion
The crystal structure of the complex between scFv
antibody 4B08 and wild-type peptide was determined at
1.35 Å resolution by the method of molecularreplacement using the coordinates of a previously
determined scFv [2]. The complex between antigen andantibody is stabilized by nine intermolecular and fourwater-mediated H-bonds, and an interaction surface of
995 ± 31 Å2 is reported. Residues Asn3 and Thr6 engage
in four intramolecular H-bonds with other residues of thepeptide in the complex (Fig. 1). Since these two residues
barely interact with the antibody, we considered them agood model to investigate the contribution of
intramolecular H-bonds in protein-peptide interactions.

Fig. 1: Intramolecular H-bonds within the peptide (wildtype) when bound to the scFv antibody 4B08 [1].
Crystal structures of 4B08 in complex with peptides
containing mutations N3A or T6A were determined at
1.96 and 1.35 Å resolution, respectively (not shown).
Each mutation individually ablate half of theintramolecular H-bonds observed above. The coordinates
and binding mode of these two peptides were essentially
identical to those of wild-type peptide. As expected themutations eliminate the intramolecular H-bonds between
their side chains (Asn3 or Thr6) and other atoms in thepeptide. Although these mutations have a significant

Photon Factory Activity Report 2017 #35 (2018)

effect in affinity (2.7 and 27-fold decrease for N3A andT6A, respectively) the structure of the bound peptide is
not altered in the crystal [1].
Structural data combined with isothermal titration
calorimetry and MD simulations lead us to propose themodel of interaction shown in Figure 2. Intramolecular Hbonds modulate the ensemble and dynamics of thepeptide in solution, bringing significant changes to theaffinity for the antibody.

Fig. 2: Model showing the influence of intramolecular Hbonds in the recognition of a peptide by an antibody [1].
In summary, our study has established a role for
intramolecular H-bonds in antibody-antigen interactions.
Acknowledgements
This work was supported by the Funding program forworld-leading Innovative R&D on Science andTechnology (FIRST) from JSPS, the Platform for Drug
Discovery, Informatics, and Structural Life Science(MEXT), and JSPS Grants-in-Aid for Scientific Research25249115 (K.T.) and 15K06962 (J.M.M.C.). We thank thestaff of the Photon Factory (Tsukuba, Japan) for excellent
technical support.
References[1] K. Miyanabe et al., J. Biochem. 164, 65 (2018).
[2] S. Kudo et al., Sci Rep. 7:39518 (2017).
* Corresponding authors: jose@phar.kyushu-u.ac.jp(JMMC), and tsumoto@bioeng.t.u-tokyo.ac.jp (KT)