Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector

https://doi.org/10.1016/j.bbrc.2018.04.045Get rights and content

Highlights

  • Crystal structure of a circularized variant of G-CSF was determined.

  • It is the first-ever crystal structure of a backbone-circularized protein.

  • Electron density proved the formation of a peptide bond connecting the ends.

  • Terminal helix was deformed to relax the stress attributed to artificial connection.

Abstract

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 310 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology.

Introduction

Backbone circularization, connecting the N- and C-termini of a polypeptide by a peptide bond, is a powerful approach for stabilizing proteins [1,2]. Since the alteration of amino acid sequence by backbone circularization (henceforth referred to as “circularization”) is much smaller 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 be especially applicable for the engineering of biopharmaceutical proteins. Among several technologies used for the generation of circularized polypeptides, the split intein-mediated circularization (often called SICLOPPS, the split-intein circular ligation of peptides and proteins) enables the connection of polypeptide termini with minimal alteration to sequences. Using that approach, only one residue (Cys or Ser) is inserted into the circularized product [3,4].

We have recently reported the design of circularized granulocyte colony-stimulating factor (G-CSF) [5]. G-CSF is a four-helix bundle cytokine that regulates the development of neutrophils. Recombinant G-CSF has been utilized as a biopharmaceutical [6]. We have designed, generated, and characterized the recombinant variants of circularized G-CSF harboring connecting segments of different lengths [5]. Comparison of the N- and C-terminal helical regions of G-CSF with the helix-turn-helix segments extracted from the structures deposited in the Protein Data Bank (PDB) suggested that two, five, and nine residues might be used to connect the G-CSF termini without distorting the protein structure. Specifically, we expected that the C163 variant harboring the shortest connector would be most stable, since a minimum and distortion-free connector should maximize the stabilizing effect by loop-shortening [1]. Nevertheless, although all variants were more stable than the linear G-CSF, C163 was less stable than the other circularized variants, perhaps because a two-residue connector was too short to allow maintenance of the original structure of G-CSF. On the other hand, C163 was intriguing as it was circularized without the formation of a linearized byproduct that typically arises from false trans-splicing reaction catalyzed by the split intein [5]. This facilitated the generation of C163 samples of high purity.

In the current report, we present the crystal structure of C163. Generation of several artificially circularized proteins has been demonstrated [[7], [8], [9]] but their crystal structures have not yet been reported, presumably because of the difficulty of obtaining sufficient quantities of pure circularized protein. We have successfully developed the C163 preparation protocol in the previous report [5] and herein crystalized the protein to understand the effect of circularization on its structure. We determined the C163 crystal structure at a resolution of 1.65 Å and confirmed that the N- and C-termini were indeed connected by a peptide bond. We further evaluated the effect of circularization on the 3D structure, by comparing with the crystal structure of linear G-CSF. Significant structural changes were apparent in the C-terminal region of C163, namely, stretch with a transition from α helix to 310 helix, and a tilt close to the N-terminal region. These structural changes suggested that the circularization forced the terminal regions to move closer to one another. In other words, the helical structure gained some flexibility to compensate for the stress associated by introducing a short connector.

Section snippets

Protein expression and purification

C163 was expressed and purified as described previously [5]. Briefly, 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, the insoluble fraction was collected, washed, resolubilized, and refolded. The refolded sample was purified by using three chromatography steps, i.e., anion-exchange chromatography on HiTrapQ (GE Healthcare),

The overall C163 structure

We determined the crystal structure of C163 at a 1.65 Å resolution (Fig. 1, Table 1). All (652) residues comprising four molecules of C163 in an asymmetric unit were modeled. Considerable structural differences between the four molecules were not apparent; the Cα atoms of these molecules superimposed with the root mean squared deviation (RMSD) of less than 0.10 Å. The connection between Gly163 and Ser1 was clearly visible in the electron density map, indicating that the structure of a

Discussion

We determined the crystal structure of C163 at a resolution of 1.65 Å. To the best of our knowledge, this is the first-ever reported crystal structure of a circularized protein. The C163 structure revealed an a priori unpredictable protein structure distortion at the connected termini.

The strategy of loop shortening improves protein stability by restricting the conformational space of the unfolded state and shifting the equilibrium toward the folded state [1]. Hence, theoretically, a shorter

Acknowledgements

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 the Japan Society for the Promotion of Science (grant no. 23510273, to S.H.).

References (24)

  • A.G.W. Leslie

    The integration of macromolecular diffraction data

    Acta Crystallogr. Sect. D Biol. Crystallogr.

    (2006)
  • P. Evans

    Scaling and assessment of data quality

    Acta Crystallogr. Sect. D Biol. Crystallogr.

    (2006)
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