Solving X-ray structure at low resolution: Natural killer cell receptor NKp30 with its tumor ligand B7-H6

T. Skálová1, O. Skořepa2, B. Kalousková2, J. Bláha2, C. Abreu2, J. Dohnálek1, O. Vaněk2

1Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Průmyslová 595, 252 50, Vestec, Czech Republic

 2Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 40 Prague, Czech Republic

t.skalova@gmail.com

 

NKp30 is an activating receptor on the surface of human natural killer (NK) cells. B7-H6 is an activating ligand expressed by various tumor cells. The crystal structure of the complex, with NKp30 protein from bacterial production and B7-H6 protein from baculovirus-infected insect cells, was published by Li et al. [1], PDB code 3PV6.

We have solved a new crystal structure of NKp30:B7-H6, in which both components come from eukaryotic cell lines (HEK293S GnTI- cells). NKp30 was used with complete glycosylation, while B7-H6 was deglycosylated after the first GlcNAc for better crystallization. The structure has been deposited in the Protein Data Bank under code 6YJP and published [2]. The structure confirmed the NKp30:B7-H6 interaction interface, as observed by Li et al. However, we observed dimers of NKp30, which are likely biologically relevant and different from dimers described previously in [3], PDB code 3NOI. The newly observed dimer of NKp30 is placed among two B7-H6 molecules in the crystal structure. This arrangement may indicate the possibility of binding of the NKp30 dimer between two B7-H6 ligands even during the contact of the NK cell and the tumor cell.

In this talk, I will focus on issues connected with the solution and refinement of the crystal structure. The data were processed to resolution 3.1 Å; however, they were anisotropic and had resolution only 4.4 Å in the weakest direction. The crystals lost diffraction during data collection, and data from several crystals were necessary to merge. Both proteins have the same immunoglobulin-like fold, so it was uneasy to place the molecules into the unit cell correctly. Refinement was performed using LORESTR (REFMAC pipeline for low-resolution structures). Electron density indicated presence of an additional very flexible protein chain; this chain was not included in the final deposited coordinates.

 

1.         Li, Y., Wang, Q., Mariuzza, R.A. (2011). J. Exp. Med. 208, 703–714.

2.         Skořepa, O., Pazicky, S., Kalousková, B., Bláha, J., Abreu, C., Ječmen, T., Rosůlek, M., Fish, A., Sedivy, A., Harlos, K., Dohnálek, J., Skálová, T., Vaněk, O. (2020). Cancers 12, 1998.

3.         Joyce, M.G., Tran, P., Zhuravleva, M.A., Jaw, J., Colonna, M., Sun, P.D. (2011) Proc. Natl. Acad. Sci. USA 108, 6223–6228.

 

This research was funded by Czech Science Foundation (18-10687S), MEYS of the Czech Republic (LTC17065, CZ.02.1.01/0.0/0.0/16_013/0001776), BIOCEV (ERDF CZ.1.05/1.1.00/02.0109), and Charles University (GAUK 927916, SVV 260427/2020). CIISB research infrastructure project LM2015043, funded by MEYS CR, is gratefully acknowledged for the financial support of experiments at the CMS. The authors also acknowledge the support and the use of Instruct-ERIC resources (PID: 1314) and iNEXT (PID: 2322) infrastructures. The Wellcome Centre for Human Genetics is supported by Wellcome Trust grant 203141/Z/16/Z. O.S. and O.V. received short-term scientific mission support from COST Action CA15126.