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.
Highlights
Perforin is a critical component of immune homeostasis, responsible for clearing virally infected cells
We have investigated the interactions of perforin C2 domain with Ca2ϩ ions by both NMR spectroscopy and x-ray crystallography, using highly soluble and stable variants of mouse perforin C2 domain in which Trp427, Tyr-430, Tyr-486, and Trp-488 were mutated to alanine
Expression and Purification of Perforin C2 Domain Mutants—Expression of the perforin C2 domain in isolation at levels required for crystallography and NMR spectroscopy has not been successful to date, despite extensive attempts over many years [17]
Summary
Perforin is a critical component of immune homeostasis, responsible for clearing virally infected cells. The second Ca2ϩ ion was coordinated by a nonconserved Asp residue (Asp-490) and found outside the CBR3 in an unusual binding position that appears to be unique to the perforin C2 domain (Fig. 1). There is no direct structural evidence of Ca2ϩ-dependent rearrangements of the CBR1 in the perforin C2 domain, and the numbers of bound Ca2ϩ ions and their binding sites remain unclear. To address these questions, we have investigated the interactions of perforin C2 domain with Ca2ϩ ions by both NMR spectroscopy and x-ray crystallography, using highly soluble and stable variants of mouse perforin C2 domain in which Trp427, Tyr-430, Tyr-486, and Trp-488 were mutated to alanine. We propose a detailed Ca2ϩ-binding mechanism of the perforin C2 domain and a role for bound Ca2ϩ in its interaction with lipid membranes
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