Photon Factory Activity Report 2019 #37 (2020)

BL-5A, BL-17A, AR-NW12A/2016G617, 2018G636

Crystal Structure of Fab Fragment of Anti-Osteocalcin
Antibody KTM219 with the Addition of Fluorophore TAMRA
Shuma YAZAKI1, Misaki KOMATSU1, Jinhua DONG2, Hiroshi UEDA2, and Ryoichi ARAI1,*1
Fac. of Tex. Sci. Tech. and Inst. for Biomed. Sci., Shinshu Univ., Ueda, Nagano 386-8567, Japan
2
Lab. for Chem. Life Sci., Tokyo Inst. of Tech., Yokohama, Kanagawa 226-8503, Japan
1 Introduction
Abe et al. developed 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]. Also, UltraQuenchbody (UQ-body), the antibody Fab fragment thatwere fluorolabeled at two of the N-terminal regions, wasdeveloped, and its improved response was observed [2].
An anti-osteocalcin C-terminal peptide antibody
KTM219 is one of the antibody Fab fragments suitablefor UQ-body. Recently, we solved a crystal structure ofthe Fab fragment of the antibody KTM219 (PDB ID:5X5X). In this study, for the purpose of further analysis
for UQ-body, a crystal was prepared with the addition ofa fluorophore TAMRA.
2 Experiments
The Fab fragment of the anti-osteocalcin C-terminal
peptide antibody KTM219 was expressed with His6-tag
in Escherichia coli, and purified by the following steps:
immobilized-metal affinity chromatography, anion
exchange chromatography, and gel filtration
chromatography. The KTM219 Fab were crystallized at20ºC with the addition of TAMRA using the hanging
drop vapor diffusion method. The KTM219 Fab andTAMRA were mixed with the same volume of reservoirsolution (0.3 M Ammonium Acetate, 0.1 M HEPES, pH
7.5, 25% PEG 3350). X-ray diffraction data werecollected at KEK Photon Factory Structural Biology
Beamline BL-5A at 95 K with 25% glycerol as a
cryoprotectant. The structure was solved by molecularreplacement method using Phaser with a model structureof KTM219 Fab (PDB ID: 5X5X). The crystal structure
was refined using COOT and REFMAC5.
3 Results and Discussion
The crystal belongs to the orthorhombic space group
P212 21, with unit cell constants of a = 66.19 Å, b = 69.52
Å, c = 96.65 Å, and contains one Fab antibody molecule
per asymmetric unit. The structure was refined to 2.14 Åresolution (Rwork = 20.5%, Rfree = 27.4%). The crystal
structure shows that the KTM219 antibody Fab fragment
comprises a light chain (VL–CL) and a heavy chain (VH–CH1) with typical immunoglobulin folds (Fig. 1A).
However, we could not find the electron density assignedto TAMRA. A deep pocket exists between VH and VLand probably provides the potential binding site for theantigen osteocalcin C-terminal peptide (BGP-C7:
RRFYGPV). There is a potential space for TAMRA to bepresent in the binding pocket. Remarkably, two loops ofVH near the binding pocket have different structurescompared to the previously solved structure (PDB ID:5X5X) of the KTM219 Fab without TAMURA (Fig. 1Band 1C). Further analysis is still needed to understandmechanisms for UQ-body in detail.

Fig. 1: Crystal structure of the Fab fragment of the antiosteocalcin antibody KTM219 with the addition ofTAMRA. (A) Ribbon representation of the overallstructure. The heavy and light chains are shown in green
and cyan, respectively. (B) Structural alignment of main
chain structures of KTM219 Fab (green/cyan) withTAMRA and KTM219 Fab (gray; PDB ID: 5X5X). (C)Closeup view of (B) around the potential binding pocket(ribbon representation). The arrows indicate the loops
that have the distinct structures.
Acknowledgements
We thank PF staff for kind assistance in X-ray
diffraction experiments. This work was supported by
JSPS KAKENHI Grants No. JP16K05841, JP17KK0104,
and JP19H02522. This work was performed under theCooperative Research Program of "NJRC Mater. &Dev."
References[1] R. Abe et al., J. Am. Chem. Soc. 133 (2011) 17386–94.
[2] R. Abe et al., Sci. Rep. 4 (2014) 4640.

*rarai@shinshu-u.ac.jp