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 Ca(2+) and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca(2+) 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 Ca(2+) binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.
authors: From the Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052.
authors: Hiromasa
authors: H
authors: Yagi
authors: the Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800.
authors: Paul J
authors: PJ
authors: Conroy
authors: From the Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052.
authors: Eleanor W W
authors: EW
authors: Leung
authors: the Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800.
authors: Ruby H P
authors: RH
authors: Law
authors: the Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, and the Departments of Microbiology and Immunology and.
authors: Joseph A
authors: JA
authors: Trapani
authors: the Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, and the Departments of Microbiology and Immunology and Genetics and Pathology, University of Melbourne, Parkville, Victoria 3052, Australia.
authors: Ilia
authors: I
authors: Voskoboinik
authors: the Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, james.whisstock@monash.edu.
authors: James C
authors: JC
authors: Whisstock
authors: From the Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, ray.norton@monash.edu.
authors: Raymond S
authors: RS
authors: Norton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D054353:Perforin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D008563:Membrane Lipids; D010767:Phosphorylcholine; D054353:Perforin; C028810:dodecylphosphocholine; D002118:Calcium
keywords: NMR; calcium-binding protein; crystal structure; immunology; multisite ligand binding; protein-membrane interaction; x-ray crystallography