Crystallization Notes
Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin

https://doi.org/10.1016/j.jsb.2015.04.015Get rights and content

Abstract

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330–450 kDa subunits, resulting in a molecular mass >3.3 MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345 nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form.

Introduction

Many molluscs possess blue blood because they use the type-3 copper-containing protein, hemocyanin, to transport oxygen. Hemocyanin is a very large multimeric glycoprotein that dissolves freely in the haemolymph. Molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330–450 kDa subunits, resulting in a molecular mass >3.3 MDa (Markl, 2013). Therefore, molluscan hemocyanins are among the largest protein structures in the world. The molluscan hemocyanin polypeptide is composed of a series of (typically 7–8) sequentially arranged paralogous functional units (FUs) of approximately 50 kDa. Ten copies of the polypeptide assemble into a cylinder of 310-Å diameter and 160-Å height. In gastropods, several cylindrical decamers further stack into didecamers or multidecamers (Markl, 2013). Each FU contains one dioxygen binding dinuclear copper site. It remains unclear why molluscs utilize such large supermolecules to transport oxygen. Molluscan hemocyanins are commonly used as adjuvants for antibody preparations, immunotherapeutic agents for the treatment of cancer, and carrier molecules for vaccines (Becker et al., 2014). These applications are based on their size and the presence of attached glycans (Geyer et al., 2005, Harris and Markl, 1999, Siddiqui et al., 2007). To further explore the molecular basis and design effective hemocyanins for applications, precise structural information has been desired for a long time. However, the unusually large size of molluscan hemocyanin has hampered the crystal structure analysis of intact molluscan hemocyanin. X-ray crystal structures have so far only been reported for a few relatively small FUs (Cuff et al., 1998, Jaenicke et al., 2010, Jaenicke et al., 2011, Perbandt et al., 2003). Consequently, structural studies on intact hemocyanin have been carried out mostly by electron microscopy (Boisset and Mouche, 2000, Gatsogiannis and Markl, 2009, Gatsogiannis et al., 2007, Gatsogiannis et al., 2015, Lamy et al., 1998, Meissner et al., 2000, Zhang et al., 2013, Zhu et al., 2014). This technique indicated that the lumen of molluscan hemocyanin cylinders is composed of a hollow cylindrical wall with several collar regions located inside the cylinder (Gatsogiannis and Markl, 2009, Gatsogiannis et al., 2007, Zhang et al., 2013, Zhu et al., 2014).

In the present study, we obtained high-quality crystals of hemocyanin from Pacific flying squids (Todarodes pacificus) possessing 8 FUs, in which hemocyanin is known to form 380-kDa decamers (Gai et al., in preparation). Negative-stain transmission electron microscopy (TEM) of these crystals demonstrated that they contain intact hemocyanin. Furthermore, we successfully obtained a full X-ray diffraction dataset at 3.0-Å resolution by merging two datasets collected from isomorphic crystals. Self-rotation function analysis indicated the presence of decameric hemocyanin with a D5 symmetry.

Section snippets

Results and discussion

Hemolymph was collected from living T. pacificus, and then individual samples were stored at 193 K until further use. Hemocyanin was the only protein observed in hemolymph by SDS–PAGE analysis (Fig. 1), and the concentration was quite high (>100 mg mL−1). Therefore, hemolymph was used for protein crystallization without purification steps. Crystallization screening was performed using commercially available kits using the sitting-drop vapor-diffusion method in which 200 nL of hemolymph was mixed

X-ray diffraction data processing

X-ray diffraction data were collected from two isomorphic crystals. Each diffraction dataset was individually indexed, integrated, and scaled with the XDS program (Kabsch, 2010). The two isomorphic datasets were merged with the XSCALE program (Kabsch, 2010) and the self-rotation function was calculated with MOLREP (Vagin and Teplyakov, 1997).

Negative-stain transmission electron microscopy of crystals

The initial crystals were washed by soaking several times into crystallization buffer, and then dissolved into a buffer containing 50-mM HEPES (pH 7.5) and

Acknowledgements

This work was supported by JSPS KAKENHI Grant number 26291008 (to Y.T.) and 25450298 (to S.K.), the Platform for Drug Discovery, Informatics, and Structural Life Science (to S.K. and Y.T.), and the Regional Innovation Strategy Support Program (to S.K., T.S., and T.Y.) of the Ministry of Education, Culture, Sports, Science, and Technology (Japan). The X-ray diffraction experiments were performed under the proposal numbers 2013G165 (Photon Factory), 2013A6829, 2013A1096, 2013B6829, 2013B1031,

Cited by (9)

  • Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand

    2019, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    Lastly, we crystallized the TpHc encapsulating the guest protein under the same crystallization conditions as the original unmodified TpHc crystal. TpHc was prepared from hemolymph extracted from Japanese flying squid as previously described [15]. Dylight488-labeled streptavidine (hereafter referred to as DyLight488-streptavidin) was purchased from Vector Laboratory (Burlingame, CA, USA).

  • Encapsulation of biomacromolecules by soaking and co-crystallization into porous protein crystals of hemocyanin

    2019, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    In these reports, a huge hollow space in the protein crystal is commonly used for encapsulation of a guest. Our previous study on the crystal structure analysis of squid hemocyanin revealed that cylindrical hemocyanins, in the crystal, stack toward the direction of the five-fold axis, which results in a linear hollow structure (Fig. 1) [14,16]. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through.

  • Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution

    2015, Structure
    Citation Excerpt :

    We hope that our crystal structure of intact hemocyanin will facilitate future developments in both bioengineering and fundamental scientific research. The procedures of crystallization and X-ray data collection have been previously described (Matsuno et al., 2015). The initial phase was determined at 10 Å resolution by molecular replacement using the PHASER program (McCoy et al., 2007) with the Cα model of the wall region of H. diversicolor hemocyanin determined by cryoelectron microscopy (PDB: 3J32) (Zhang et al., 2013) as a search model.

  • Molluscan Hemocyanins

    2020, Subcellular Biochemistry
View all citing articles on Scopus
View full text