Volume 62, Issue 1, Pages 21-33 (April 2016) Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses  Santosh Kumar, Sumati Rajagopalan, Pabak Sarkar, David W. Dorward, Mary E. Peterson, Hsien-Shun Liao, Christelle Guillermier, Matthew L. Steinhauser, Steven S. Vogel, Eric O. Long  Molecular Cell  Volume 62, Issue 1, Pages 21-33 (April 2016) DOI: 10.1016/j.molcel.2016.03.009 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Zinc-Induced Polymerization of Purified Ectodomain of KIR2DL1, Residues 1–224, into Filaments (A) Intrinsic Trp fluorescence spectra. 1.5 μM soluble KIR2DL1 was treated with no zinc (blue) or 5 μM (red), 12.5 μM (green), 20 μM (dotted blue), and 50 μM (dotted red) ZnCl2 for 30 min. (B) Dependence of Trp fluorescence at 338 nm (I338 nm) on ZnCl2 concentration. Error bars represent SD of the mean determined from three independent experiments. (C) Intrinsic Trp fluorescence spectra of KIR2DL1 in supernatant after centrifugation. 1.5 μM KIR2DL1 was treated with no zinc (blue), 50 μM ZnCl2 for 30 min (red), or 50 μM ZnCl2 for 30 min followed by 100 μM EDTA for 30 min (black). (D) TEM images after treatment of 1.5 μM KIR2DL1 with 50 μM ZnCl2 for 30 min. (E and F) TEM images after treatment of 1.5 μM KIR2DL1 with 50 μM ZnCl2 (E) or 50 μM ZnCl2 followed by 100 μM EDTA (F). (G) AFM image after treatment of 1.5 μM soluble KIR2DL1 with 50 μM ZnCl2 for 30 min. (H) Intrinsic Trp fluorescence spectra of native (dotted black) and urea-induced unfolded (dotted green) KIR2DL1. (I) Kinetics of refolding in the absence (black) and presence of 5 μM (red), 10 μM (solid blue), and 50 μM (dotted blue) ZnCl2. Dotted black and dotted green lines represent signals for native and unfolded receptor, respectively. (J) Intrinsic Trp fluorescence spectra of unfolded KIR2DL1 (dotted green) and of the end products of KIR2DL1 refolding in the absence of zinc (black) and in the presence of 5 μM (red), 10 μM (solid blue), and 50 μM (dotted blue) ZnCl2. In (H)–(J), the final concentration of KIR2DL1 was 1.5 μM. See also Figure S1. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Zinc-Induced KIR2DL1 Filament Formation on NK Cells (A and B) Steady-state anisotropy measurements in 96-well plate mode. 400 μM ZnCl2 and 1 mM EDTA were added at the indicated times to YTS-2DL1-Venus cells (A) or YTS–GFP-2DL1 cells (B). Error bars represent SD of the mean determined from three (A) and seven (B) independent experiments. (C) Steady-state anisotropy measurements in imaging mode. YTS-2DL1-Venus cells were treated with 50 μM or 100 μM ZnCl2, as indicated, for 1.5 hr. Anisotropy (top panels) and fluorescence intensity (bottom panels) images are shown. (D) Plot of median anisotropy against fluorescence intensity for every pixel in (C). (E–G) TEM images of KIR2DL1 immunoprecipitated from YTS-2DL1-Venus cells that were incubated with no zinc (E), 50 μM ZnCl2 (F), or 100 μM ZnCl2 (G) for 1.5 hr. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 The N-Terminal HExxH Motif Is Required for Zinc-Induced Polymerization of KIR2DL1 into Filaments (A) Intrinsic Trp fluorescence spectra of 1.5 μM soluble KIR2DL1-H1,5A treated with no zinc (blue) or 5 μM (red), 12.5 μM (green), 20 μM (dotted blue), 30 μM (black), and 50 μM (dotted red) ZnCl2 for 30 min. (B) Dependence of Trp fluorescence at 338 nm (I338 nm) on ZnCl2 concentration for wild-type KIR2DL1 (circles) and KIR2DL1-H1,5A (squares). Error bars represent SD of the mean determined from three independent experiments. (C) TEM images of soluble KIR2DL1-H1,5A treated with 50 μM ZnCl2 for 30 min. (D) Anisotropy measurements on YTS–2DL1-H1,5A-Venus cells treated as in Figure 2. Error bars represent SD of mean determined from three independent experiments. (E and F) TEM images of KIR2DL1-H1,5A immunoprecipitated from YTS-2DL1-H1,5A-Venus cells treated for 1.5 hr in the absence (E) or presence (F) of 200 μM ZnCl2. See also Figure S2. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 The Stem Region of KIR2DL1 Is Required for Zinc-Induced Polymerization into Filaments on Cells and for Inhibitory Function (A) Intrinsic Trp fluorescence spectra of 1.5 μM soluble KIR2DL1-SD treated with no zinc (blue) or 5 μM (red), 12.5 μM (green), 20 μM (dotted blue), and 50 μM (dotted red) ZnCl2. (B) Dependence of the wavelength of maximum fluorescence (λmax) on ZnCl2 concentration. (C) TEM images of soluble KIR2DL1-SD treated with 50 μM ZnCl2 for 30 min. (D and E) TEM images of KIR2DL1-SD immunoprecipitated from YTS-2DL1-SD-Venus cells treated in the absence (D) or presence (E) of 100 μM ZnCl2 for 1.5 hr. (F) Scattering intensity of KIR2DL1 immunoprecipitated from YTS-2DL1-Venus (WT) and YTS-2DL1-SD-Venus (SD) cells treated in the absence or presence of 100 μM ZnCl2, as indicated, for 1.5 hr. The value of scattering intensity (SI) obtained in the absence of exogenous zinc was set to 1. Error bars represent SE of the mean determined from four independent experiments. ∗∗p < 0.005. Statistical analysis was conducted by non-parametric t test using GraphPad Prism. (G) Staining with anti-HLA-C Ab F4-326 of 221-HLA-Cw4-ICP47 cells that had been incubated with increasing amounts of an HLA-Cw4-specific peptide for 16 hr at 26°C. MFI, mean fluorescence intensity. (H) Lysis of peptide-loaded 221-HLA-Cw4-ICP47 cells by YTS-2DL1-Venus (circles), YTS-2DL1-H1,5A-Venus (triangles), and YTS-2DL1-SD-Venus (squares) cells. Lysis of target cells in the absence of exogenous peptide was set to 1. Error bars represent SD from three independent experiments. See also Figure S3. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 High-Resolution Imaging of Whole Cells after KIR2DL1 Expression and Treatment with ZnCl2 (A) SEM images of HEK293T cells stably transfected with KIR2DL1 (293T-2DL1) that had been treated with no ZnCl2 or 200 μM ZnCl2 for 2 hr. The rightmost panel is a higher magnification of membrane protrusions. Scale bars, 5 μm in the first two panels and 500 nm in the third panel. (B) SEM images of HEK293T cells that had been either mock-transfected or transfected with KIR2DL1, as indicated, and treated with 200 μM ZnCl2. The rightmost panel is an enlargement of the middle panel. Scale bars, 5 μm in the first two panels and 1 μm in the third panel. (C) NanoSIMS images of HEK293T cells that were either untransfected or stably transfected with KIR2DL1 (293T-2DL1) and treated with the indicated ZnCl2 concentrations for 2 hr. Mass images, as determined by CN− emission-based pixel intensities, are shown. Scale bars, 5 μm. (D) Higher resolution NanoSIMS images of membrane protrusions on 293T-2DL1 cells. CN− (leftmost panel) and ZnO− (middle and rightmost panels) images were acquired simultaneously. In the rightmost panel, ZnO− counts within the indicated area (yellow outline) are pseudocolored red. Scale bars, 2 μm. (E) The mean ZnO− emission values obtained from the filopodia-like protrusions on 293T-2DL1 cells, treated with 50 μM and 200 μM ZnCl2, were normalized relative to those obtained from adjacent regions of the silicon support (background). Error bars represent SEM obtained from the analysis of multiple cells for each of the ZnCl2 concentrations. ∗p < 0.05. GraphPad Prism was used to perform the t test. (F) ZnO−-CN− ratio of cell edges and protrusions on 293T-2DL1 cells that were treated with 50 μM and 200 μM ZnCl2. The ZnO−-CN− ratios were normalized relative to randomly selected areas in the cell body. Error bars represent SEM obtained from the analysis of multiple cells for each of the ZnCl2 concentrations. ∗p < 0.05. A non-parametric Mann-Whitney test was used to compare the values for protrusions to those for the corresponding cell edge. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Zinc-Induced Polymerization Is Not Sufficient for KIR2DL1 Tyrosine Phosphorylation and Causes Loss of Ab and HLA-C Binding (A–C) YTS-2DL1-Venus and NKL-2DL1-SBP cells were treated sequentially with 200 μM ZnCl2 for 1.5 hr and pervanadate (Perv.) for 5 min. Cells receiving a single treatment or no treatment are indicated below each lane. Immunoprecipitated samples were immunoblotted for phospho-Tyr (p-Tyr). (A and B) KIR2DL1-Venus was immunoprecipitated with an anti-GFP nanobody (A) or an Ab to SHP-1 (B) from YTS-2DL1-Venus cells treated with ZnCl2 and 200 μM pervanadate, as indicated. The position of KIR2DL1-Venus and SHP-1 in (B) is indicated by one and two asterisks, respectively. (C) KIR2DL1-SBP (SBP) was pulled down with streptavidin beads from NKL-2DL1-SBP cells treated with ZnCl2 and 800 μM pervanadate, as indicated. Recovery in the pull-downs was monitored by immunoblotting for the 2A C-terminal tag. IP, immunoprecipitation. (D and E) YTS-2DL1-Venus cells were stained with anti-KIR2DL1 mAb (clone EB6) coupled with allophycocyanin after treatment with no zinc (circles), 100 μM ZnCl2 (squares), or 200 μM ZnCl2 (triangles) for the indicated time. (E) Venus fluorescence was acquired simultaneously on the same samples shown in (D). The data shown in (D) and (E) are representative of two independent experiments. (F) YTS-GFP-2DL1 cells were treated with ZnCl2 for 1.5 hr and stained with anti-KIR2DL1 mAb (clone 143211) (circles) or anti-GFP Ab (squares). (G) YTS-2DL1-Venus cells were treated with ZnCl2 for 1.5 hr and stained with Abs to KIR2DL1 (clone EB6) (circles), receptor 2B4 (triangles), CD28 (inverted triangles), and MHC class I (diamonds). Abs were directly coupled with phycoerythrin. (H) YTS-2DL1-Venus cells were stained with anti-KIR2DL1 mAb (clone 143211) either before (squares) or after (circles) treatment with ZnCl2 for 1.5 hr. An anti-IgG Ab was used to detect the primary Ab. (I) YTS-2DL1-Venus cells were stained with anti-KIR2DL1 mAb (EB6-PE) (squares), and with HLA-Cw4 tetramer coupled to Alexa Fluor 647 (triangles) after treatment with the indicated concentrations of ZnCl2 for 2 hr. Venus fluorescence was acquired at the same time (circles). (J) NKL-2DL1-SBP cells were treated and stained as in (I), with the addition of an anti-CD94-FITC (fluorescein isothiocyanate) Ab (circles). Error bars represent SD of the mean determined from three independent experiments. See also Figure S4. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 KIR2DL1 Filamentous Polymers at Functional Inhibitory NK-Target Cell Synapses (A and B) TEM images (A) and AFM images (B), in topography mode, of KIR2DL1 immunoprecipitated from YTS-2DL1-Venus cells that had been mixed with activating 221-HLA-Cw3 and inhibitory 221-HLA-Cw15 target cells. Scale bars, 100 nm in (A) and 330 nm in (B). (C) TEM images of KIR2DL1 pulled down with streptavidin beads from NKL-2DL1-SBP cells that had been mixed with activating 221 and inhibitory 221-HLA-Cw15 target cells. The top rows show images from one experiment, and the rest are images from a separate experiment. The bottom row shows images of KIR2DL1 pull-down samples treated with 25 μM TPEN for 30 min or 100 μM EDTA for 30 min, as indicated. Scale bars, 100 nm. Molecular Cell 2016 62, 21-33DOI: (10.1016/j.molcel.2016.03.009) Copyright © 2016 Elsevier Inc. Terms and Conditions