Photon Factory Activity Report 2017 #35 (2018)

BL-1A, AR-NW12A/2015G059

Crystal structure of the backbone-circularized G-CSF
Takamitsu MIYAFUSA1, Risa SHIBUYA2, and Shinya HONDA1, 2*
Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8561, Japan2
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences,
The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
1

1 Introduction
Backbone circularization, connecting the N- and Ctermini of a polypeptide by a peptide bond, is a powerfulapproach for stabilizing proteins. Since the alteration ofamino acid sequence by backbone circularization(henceforth referred to as “circularization”) is muchsmaller than that introduced by other stabilization
strategies (e.g., amino acid substitution), circularization is
also much less likely to induce immunogenicity.
Therefore, we propose that the method might beespecially applicable for the engineering ofbiopharmaceutical proteins.
We have recently reported the design of circularized
granulocyte colony-stimulating factor (G-CSF) [1]. GCSF is a four-helix bundle cytokine that regulates thedevelopment of neutrophils. Recombinant G-CSF has
been utilized as a biopharmaceutical.
In this study, we present the crystal structure of acircularized variant of the granulocyte colony-stimulating
factor (G-CSF), C163. The variant was created byconnecting N- and C- terminal helical segments with a
two-residue connector.
2 Experiment
C163 harboring the split intein from Nostoc
punctiforme was expressed in Escherichia coli BL21
(DE3) cells. Circularization of C163 by the split intein
occurred in the bacterial cells. Upon cell sonication, theinsoluble fraction was collected, washed, resolubilized,and refolded. The refolded sample was purified by using
three chromatography steps, i.e., anion-exchangechromatography on HiTrapQ (GE Healthcare), sizeexclusion chromatography on Superdex 75 (GEHealthcare), and anion-exchange chromatography onMonoQ (GE Healthcare).
C163 was concentrated to 7 mg/mL and used incrystallization trials. After screening using Crystal
Screen (HAMPTON RESEARCH), suitable crystals wereobtained in 0.4 M ammonium phosphate buffer.
3 Results and Discussion
We determined the crystal structure of C163 at a 1.65Å resolution (Figure 1, PDB entry code: 5GW9). The
connection between Gly163 and Ser1 was clearly visible
in the electron density map, indicating that the structureof a circularized protein was indeed determined. Most
parameters for the peptide bond geometry between
Gly163 and Ser1 were almost the same as the ideal values

from the International Tables Online site hosted by theInternational Union of Crystallography [2].

Figure 1 Crystal structure of C163.
To the best of our knowledge, this is the first-everreport of a crystal structure of a backbone-circularized
protein. Until now, only one published report exists on
the 3D structure of circularized proteins, in which thecircularized variants of the N-SH3 domain from proteinc-Crk were evaluated by NMR analysis [3]. In that study,
the peptide bond between N- and C- termini was createdby applying additional constraints, thus our crystal
structure allows the first-ever direct observation of thecircularized segment.
Our observations will further the development ofmethodology to stabilize the structure of proteins forbiomedical and other applications.
Acknowledgement
We thank members of the Photon Factory (Tsukuba,Japan) for their assistance during X-ray data collection.
This work was supported in part by a grant from theJapan Society for the Promotion of Science (grant no.
23510273, to S.H.).
References[1] T. Miyafusa et al., ACS Chem. Biol. 12, 10 (2017).
[2] R.A. Engh and R. Huber, International Tables for
Crystallography. Vol. F, Chapter 18.3 (2006).
[3] Schumann et al., Frontiers in Chemistry, 3, 26 (2015)
Research Achievements
1. T. Miyafusa et al., Biochem. Biophys. Res. Commun.,(2018) Epub ahead of print.
* s.honda@aist.go.jp