The C-type lectin-like receptor Nkrp1b: Structural proteomics reveals features affecting protein conformation and interactions

0508013 - MBÚ 2020 RIV NL eng J - Journal Article
Hernychová, Lucie - Rosůlek, Michal - Kádek, Alan - Mareška, V. - Chmelík, Josef (ed.) - Adámková, Ljubina - Grobárová, Valeria - Šebesta, O. - Kukačka, Zdeněk - Skála, Kristián - Spiwok, V. - Černý, J. - Novák, Petr
The C-type lectin-like receptor Nkrp1b: Structural proteomics reveals features affecting protein conformation and interactions.
Journal of Proteomics. Roč. 196, MAR 30 (2019), s. 162-172. ISSN 1874-3919. E-ISSN 1876-7737
R&D Projects: GA ČR(CZ) GA16-24309S; GA MŠMT(CZ) LQ1604; GA MŠMT(CZ) LM2015043; GA MŠMT(CZ) ED1.1.00/02.0109
Research Infrastructure: Czech-BioImaging - 90062
Institutional support: RVO:61388971
Keywords : Structural mass spectrometry * Chemical cross-linking * Ion mobility
OECD category: Microbiology
Impact factor: 3.509, year: 2019
Method of publishing: Limited access
https://www.sciencedirect.com/science/article/pii/S187439191830397X?via%3Dihub

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo.

Significance: Despite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.
Permanent Link: http://hdl.handle.net/11104/0298974