Photon Factory Activity Report 2014 #32 (2015) B

BL-5A, AR-NE3A, AR-NW12A/2012G748, 2014G673

Crystal Structure of Fab Fragment of an Anti-Osteocalcin C-Terminal Peptide
Antibody KTM219
Misaki Komatsu1, Jinhua Dong2, Hiroshi Ueda2,§ and Ryoichi Arai1,*1
Fac. of Tex. Sci. Tech., Shinshu Univ., Ueda, Nagano 386-8567, Japan
2
Chemical Resources Lab., Tokyo Inst. of Tech., Yokohama, Kanagawa 226-8503, Japan
1 Introduction
Recently, we described a novel reagentless fluorescentbiosensor strategy named Quenchbody (Q-body), whichfunctions via the antigen-dependent removal of thequenching effect on a fluorophore that is attached to asingle-chain antibody variable region [1]. More recently,
to explore the practical utility of Quenchbodies, wedeveloped “Ultra-Quenchbody (UQ-body),” antibody Fabfragments that were fluorolabeled at two of the Nterminal regions, and an improved response due toenhanced quenching via dye-dye interactions wasobserved. An anti-osteocalcin C-terminal peptide
antibody KTM219 is one of the antibody Fab fragments
suitable for UQ-body. To analyze the detailed structural
mechanism of UQ-body and utilize it for further antibody
engineering, we have solved the crystal structure of theFab fragment of an anti-osteocalcin C-terminal peptide
antibody KTM219.
2 Experiments
The Fab fragment of anti-osteocalcin C-terminal
peptide antibody KTM219 was expressed with His6 tag inEscherichia coli, and purified by the following steps:
immobilized-metal affinity chromatography, anion
exchange
chromatography,andgel
filtration
chromatography. The KTM219 Fab was crystallized at20ºC using the hanging drop vapor diffusion method. TheKTM219 Fab (1 μL) was mixed with the same volume ofreservoir solution (0.6 M sodium formate, 29% PEG3350). X-ray diffraction data collections were performedat KEK Photon Factory Structural Biology Beamlines at
95 K with reservoir solution added to 25% PEG 400 as acryoprotectant. The structure was solved by molecularreplacement method using Phaser with a model structureof anti-emmprin antibody 4A5 Fab (PDB: 4KUZ) [3].
The crystal structure was refined using COOT andREFMAC5.
3 Results and Discussion
The KTM219 Fab crystal belongs to the orthorhombic
space group P212 21, with unit cell constants of a = 64.80
Å, b = 71.47 Å, c = 96.88 Å, and contains one Fabantibody molecule per asymmetric unit. The structure wasrefined to 1.9 Å resolution (Rwork = 19.1%, Rfree = 24.2%).
The crystal structure shows that the KTM219 antibody
Fab fragment comprises a light chain (VL – CL) and aheavy chain (VH–CH1) with typical immunoglobulin folds(Fig. 1). A deep pocket forms between VH and VL, and it
may provide the binding site for the antigen osteocalcin
C-terminal peptide (BGP-C7: RRFYGPV). We speculatethat the C-terminus of the antigen peptide is inserted into
the pocket and the two Arg residues of the antigen may berecognized by the acidic Asp residues on the edge of theentrance of the binding pocket. The present structure of
the KTM219 Fab provides useful structural information.
To understand the antigen recognition and UQ-bodymechanisms of KTM219 in detail, further study iscurrently in progress.

Fig. 1. The crystal structure of the Fab fragment of ananti-osteocalcin C-terminal peptide antibody KTM219.
The heavy and light chains are shown in green and cyan,
respectively.
Acknowledgements
We thank the PF staff for assistance in X-ray
diffraction experiments. This work was supported by
JSPS KAKENHI Grant No. 24780097.
References[1] R. Abe et al., J. Am. Chem. Soc. 133 (2011) 17386–94.
[2] R. Abe et al., Sci. Rep. 4 (2014) 4640.
[3] A. Teplyakov et al., Proteins 82 (2014) 1679–83.

*rarai@shinshu-u.ac.jp§ueda@res.titech.ac.jp