Photon Factory Activity Report 2016 #34 (2017)

BL-17A/2015G043

Development of a novel antibody fragment format “Fv-clasp”
1

Takao Arimori1 and Junichi Takagi1,*Osaka University, Suita, Osaka 565-0871, Japan

1 Introduction
Antibody fragments such as Fab and single-chain Fv(scFv) fragments have been widely used as research tools.
In the structural biology field, antibody fragments areused as “crystallization chaperone” to aid crystallizationof crystallization-resistant proteins. So far, many crystal
structures of biologically and medically important
proteins have been determined by using Fab or scFv
fragments [1, 2]. However, each of these antibody
fragments has potential problems to use as crystallization
chaperon. For example, it is known that Fab fragment has
structural flexibility between variable (Fv: VH, VL) andconstant (CH1, CL) domains, which may contributenegatively to crystallization [3]. ScFv lacks the constantdomains, resulting in instability of Fv region, and hence,some antibodies are not compatible with the scFv format.
To overcome these problems, we designed a novel
antibody fragment format termed “Fv-clasp”. In the Fvclasp format, SARAH domain derived from human Mst1
protein that forms an antiparallel coiled-coil structure wasfused with C-termini of VH and VL to stabilize Fvfragment (Fig. 1). In addition, for further stabilization of
the heterodimer conformation, we introduced Cys
residues into the SARAH domains to form an inter-chain
disulfide bond.
To know whether Fv-clasp assumes the expectedstructure, we tried to determine the crystal structure ofFv-clasp derived from 12CA5, a commercially available
anti-HA tag antibody.
2 Experiment
VH and VL domains of 12CA5 were fused with theSARAH domain from human Mst1 via a Gly-Ser linker.
These chimeric proteins were separately produced in E.
coli cells and were expressed as inclusion bodies. Afterthe solubilization of the inclusion bodies with 6 M
guanidine hydrochloride, VH-SARAH and VL-SARAH
were mixed with each other, and 12CA5 Fv-clasp wasrefolded using a stepwise dilution method. The refolded
sample was purified by size-exclusion and anionexchange chromatographies. For crystallization trial,
12CA5 Fv-clasp was concentrated to approximately 8
mg/mL and mixed with antigen peptide (HA peptide) at amolar ratio of 1 : 5.
A total of 288 conditions were screened forcrystallization using commercially available screening
kits, and after a few days, crystals had appeared in 51
conditions. However, as a result of preliminary X-ray
diffraction experiment for the several initial crystals, they
all showed poor diffraction quality. To produce crystals
suitable for X-ray analysis, crystallization conditions
were optimized, and diffraction quality crystals wereobtained under the condition of 0.1 M Na/K phosphate(pH6.2) and 1.4 M ammonium sulfate.
X-ray diffraction data were obtained at the beam line
BL-17A of Photon Factory. The data was processed andscaled to maximum resolution of 3.0 Å using HKL2000[4]. The crystal belonged to space group C2 andcontained one 12CA5 Fv-clasp molecule in theasymmetric unit. Initial phase was determined bymolecular replacement analysis with PHASER [5] using
crystal structures of an Fab fragment of mouse IgG (PDBID: 1HIL) and the Mst1 SARAH domain (PDB ID:4NR2) as search models. Structural model was refinedwith REFMAC 5 [6] and the model was modified withCOOT [7].
3 Results and Discussion
Overall structure of the 12CA5 Fv-clasp is shown inFig. 2. The SARAH domains formed disulfide-bridged
anti-parallel coiled-coil structure at the bottom of the Fv
opposite from the antigen binding site as our designconcept shown in Fig. 1. VH and VL domains of 12CA5Fv-clasp formed a heterodimer with a generalconfiguration observed in many antibody structures.
Electron density attributable to the antigen peptide wasobserved in the antigen binding cleft between the VH andVL domains, and all 9 residues of HA peptide could be
assigned in the map (Fig. 2). The HA peptide wasspecifically recognized by 12CA5 Fv-clasp throughextensive hydrogen bonding and hydrophobic interactions.
These facts indicate that, although crystal structure of
12CA5 has not been reported to date, our structure
represents genuine antigen-binding conformation of theantibody. Furthermore, formation of the functional
structure of the Fv region demonstrated that the fusion of
the SARAH domains did not affect the conformation ofthe Fv region.
The crystal structure of 12CA5 Fv-clasp illustrated that
our attempt to develop a novel antibody fragment using
the SARAH domain was successful. However, it still has
a problem, because diffraction powers of the crystals
were not enough to be used as crystallization chaperones
even after the optimization of the crystallization condition.
Therefore, further optimization of Fv-clasp design must
be sought. Structural information of 12CA5 Fv-clasp
obtained here will help us to re-design of Fv-clasp.

Photon Factory Activity Report 2016 #34 (2017)

Fig. 1 Design of Fv-clasp

Fig. 2 Crystal structure of 12CA5 Fv-clasp
Acknowledgement
We would like to thank the staff of the beamlines at
Photon Factory for providing data-collection facilities andfor support.
References[1] Koide, S., Engineering of recombinant crystallization
chaperones. Curr. Opin. Struct. Biol., 2009. 19(4): p.
449-457.
[2] Griffin, L. and A. Lawson, Antibody fragments astools in crystallography. Clin. Exp. Immunol., 2011.
165(3): p. 285-291.
[3] Stanfield, R.L., et al., Antibody elbow angles are
influenced by their light chain class. J. Mol. Biol.,2006. 357(5): p. 1566-1574.
[4] Otwinowski, Z. and W. Minor, Processing of X-ray
Diffraction Data Collected in Oscillation Mode.
Methods Enzymol., 1997. 276: p. 307-326.
[5] Mccoy, A.J., et al., Phaser crystallographic software.
J. Appl. Cryst., 2007. 40: p. 658-674.
[6] Murshudov, G.N., et al., REFMAC5 for the refinementof macromolecular crystal structures. Acta.
Crystallogr. D. Biol. Crystallogr., 2011. 67: p. 355367.
[7] Emsley, P., et al., Features and development of Coot.
Acta. Crystallogr. D. Biol. Crystallogr., 2010. 66(Pt4): p. 486-501.
* takagi@protein.osaka-u.ac.jp