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

L. Hernychová, M. Rosůlek, A. Kádek, V. Mareška, J. Chmelík, L. Adámková, V. Grobárová, O. Šebesta, Z. Kukačka, K. Skála, V. Spiwok, J. Černý, P. Novák,

. 2019 ; 196 (-) : 162-172. [pub] 20181108

Jazyk angličtina Země Nizozemsko

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc20022828

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.

Citace poskytuje Crossref.org

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$a Hernychová, Lucie $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic. Electronic address: lucka.hernychova@gmail.com.
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$a 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.
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$a Rosůlek, Michal $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Kádek, Alan $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Mareška, Václav $u Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic.
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$a Adámková, Ljubina $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Grobárová, Valéria $u Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Šebesta, Ondřej $u Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Skála, Kristián $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic.
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$a Spiwok, Vojtěch $u Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic.
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$a Černý, Jan $u Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic.
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$a Novák, Petr $u BIOCEV- Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic. Electronic address: pnovak@biomed.cas.cz.
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