Structural basis for Ca2+-mediated interaction of the perforin C2 domain with lipid membranes

Hiromasa Yagi, Paul J Conroy, Eleanor W W Leung, Ruby H P Law, Joseph A Trapani, Ilia Voskoboinik, James Whisstock, Raymond S Norton

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Abstract

Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with do decylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.
Original languageEnglish
Pages (from-to)25213-25226
Number of pages14
JournalJournal of Biological Chemistry
Volume290
Issue number42
DOIs
Publication statusPublished - 2015

Cite this

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title = "Structural basis for Ca2+-mediated interaction of the perforin C2 domain with lipid membranes",
abstract = "Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with do decylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.",
author = "Hiromasa Yagi and Conroy, {Paul J} and Leung, {Eleanor W W} and Law, {Ruby H P} and Trapani, {Joseph A} and Ilia Voskoboinik and James Whisstock and Norton, {Raymond S}",
year = "2015",
doi = "10.1074/jbc.M115.668384",
language = "English",
volume = "290",
pages = "25213--25226",
journal = "Journal of Biological Chemistry",
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publisher = "American Society for Biochemistry and Molecular Biology",
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Structural basis for Ca2+-mediated interaction of the perforin C2 domain with lipid membranes. / Yagi, Hiromasa; Conroy, Paul J; Leung, Eleanor W W; Law, Ruby H P; Trapani, Joseph A; Voskoboinik, Ilia; Whisstock, James; Norton, Raymond S.

In: Journal of Biological Chemistry, Vol. 290, No. 42, 2015, p. 25213-25226.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Structural basis for Ca2+-mediated interaction of the perforin C2 domain with lipid membranes

AU - Yagi, Hiromasa

AU - Conroy, Paul J

AU - Leung, Eleanor W W

AU - Law, Ruby H P

AU - Trapani, Joseph A

AU - Voskoboinik, Ilia

AU - Whisstock, James

AU - Norton, Raymond S

PY - 2015

Y1 - 2015

N2 - Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with do decylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.

AB - Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with do decylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.

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