The amyloid structure of mouse RIPK3 (receptor interacting protein kinase 3) in cell necroptosis

Abstract

RIPK3 amyloid complex plays crucial roles during TNF-induced necroptosis and in response to immune defense in both human and mouse. Here, we have structurally characterized mouse RIPK3 homogeneous self-assembly using solid-state NMR, revealing a well-ordered N-shaped amyloid core structure featured with 3 parallel in-register β-sheets. This structure differs from previously published human RIPK1/RIPK3 hetero-amyloid complex structure, which adopted a serpentine fold. Functional studies indicate both RIPK1-RIPK3 binding and RIPK3 amyloid formation are essential but not sufficient for TNF-induced necroptosis. The structural integrity of RIPK3 fibril with three β-strands is necessary for signaling. Molecular dynamics simulations with a mouse RIPK1/RIPK3 model indicate that the hetero-amyloid is less stable when adopting the RIPK3 fibril conformation, suggesting a structural transformation of RIPK3 from RIPK1-RIPK3 binding to RIPK3 amyloid formation. This structural transformation would provide the missing link connecting RIPK1-RIPK3 binding to RIPK3 homo-oligomer formation in the signal transduction.

Fig. 1. Alignment of RHIM sequence from various proteins and two published structures containing of RHIM.

a Alignment of RHIM sequence of human (h), mouse (m), rat (r), drosophila (dr), and herpesviruses (M45, ICP6) proteins and prion-forming domain of P. anserine proteins (Het-s1, Het-s2). The most conserved tetrad sequence is highlighted at the center. The alignment was performed by clustalw2 online software. Receptor interacting protein kinase 1 (RIPK1), toll-interleukin-1 receptor domain-containing adapter protein inducing interferon beta (TRIF), and DNA-dependent activator of interferon-regulatory factors (DAI) are three proteins found in necroptosis pathway. b The SSNMR structures of human RIPK1/RIPK3 hetero-amyloid (PDB:5V7Z) and Het-s fibrils (PDB:2KJ3).

Fig. 2. The EM, X-ray diffraction, and AFM images of mouse RIPK3 fibrils.

a The domain components of the full-length mouse RIPK3. b Protein sequence of mouse RIPK3 construct used for structural elucidation. c Electron micrograph of amyloid fibrils (scale bar 200 nm). Mouse RIPK3 fibrils have the straight, unbranched appearance of typical amyloid fibrils. The picture on the right is an expanded view of the boxed area on the left image. The experiment was repeated for more than 5 times. d The X-ray diffraction of mouse RIPK3 fibrils. The blue arrow indicated equatorial and meridional reflection at about 9.7 Å and 4.7 Å resolutions, respectively. The experiment was repeated for 3 times. e The AFM image of mouse RIPK3 fibrils on mica surface. f The height profile at three positions of mouse RIPK3 fibrils corresponding to the three positions indicated by the arrows in (e), showing fibril diameter about 1.8 ± 0.2 nm. g One BT-TEM image of mouse RIPK3 fibrils, with tobacco mosaic virus (TMV) particles as standards for MPL measurement. h MPL histogram of mouse RIPK3 fibrils derived from BT-TEM images. Vertical red line indicated MPL value of 20.3 kDa/nm, the expected value if a single molecule lies in the cross-β unit of the fibril. The variations in the background intensity is analyzed in Supplementary Fig. 2. Figure 1e was a representative image of nine images from three sample preparations and BT-TEM in Fig. 1g was a representative image of more than 30 images from about ten sample preparations.

Fig. 3. SSNMR spectra of uniformly labeled mouse RIPK3 fibrils.

2D ¹³C–¹³C (a), 2D ¹³C–¹⁵N NcaCX (b), and 2D ¹³C–¹⁵N NcoCX (c) with 50 ms DARR mixing. The experiments were carried on a Bruker 700 MHz MAS NMR spectrometer with ω_r = 15 kHz, T = 303 K, and 83.33 kHz ¹H decoupling field applied during acquisition.

Fig. 4. Secondary structure prediction from the assigned chemical shifts.

a Secondary structure prediction of mouse RIPK3 construct with PSIPRED. b Plot of the difference in the secondary chemical shift between Cα and Cβ, a negative value indicative of β-sheet secondary structures. c Predicted protein dihedral angles φ and ψ using TALOS-N based on SSNMR chemical shifts. Data are presented as mean values ± SD from predicted dihedral angles φ and ψ values of 25 best structures.

Fig. 5. SSNMR spectra of uniformly and sparsely ¹³C-labeled mouse RIPK3 fibrils highlighting some long-range inter-residue correlation peaks.

a 2D ¹³C–¹³C correlation spectrum of sparsely ¹³C-labeled mouse RIPK3 fibrils using [2-¹³C]-labeled glycerol with 200 ms DARR mixing, showing the carbonyl region. The long-range correlation peak L456Cβ/Cγ-Q449Cδ are highlighted. b 2D ¹³C–¹³C correlation spectrum of uniformly ¹³C-labeled mouse RIPK3 fibrils with 500 ms DARR mixing, showing the aromatic region. The dashed lines indicate the correlation peaks of F442, and the solid lines indicate the correlation peaks of Y453. c ¹³C–¹⁵N TEDOR correlation spectrum of sparsely ¹³C-labeled mouse RIPK3 fibrils using [2-¹³C]-labeled glycerol with 6.4 ms z-filtered TEDOR recoupling time. The protein is also uniformly ¹⁵N-labeled. TEDOR shows the correlation peaks of N454Nδ2-G451Cα and Q449Nε2-L456Cγ. d 2D ¹³C–¹³C correlation spectrum of sparsely ¹³C-labeled mouse RIPK3 fibrils using [1,3-¹³C]-labeled glycerol with 500 ms DARR mixing. The assignment of V441Cγ-G451Cα is unambiguous.

Fig. 6. Structural model of mouse RIPK3 fibril core (PDB ID 6JPD).

a Superposition of ten monomer conformations with the lowest energy, calculated using XPLOR-NIH software. The dimension of the structure is labeled on the sides. The unambiguous constraints used in the calculation are also marked using the dashed lines. b Stick representation of the mouse RIPK3 fibril medoid model selected from 10 fibril structures with the lowest energy. Both (a, b) are viewed down the fibril axes. c Side view of medoid model using cartoon representation. d Side view of medoid model indicating possible hydrogen bonding between sides chains of N443, N444, Q449, and N452 (The labels in the figure are ASN3, ASN4, GLN9, and ASN12). All figures were prepared using VMD (https://www.ks.uiuc.edu/Research/vmd/).

Fig. 7. Cell-based functional assay.

a The monomer structure of mouse RIPK3, showing the three β-strand segments in red and three single mutation sites. b Mutation of F442, Q449, or L456 to D, or quadruple alanine mutations of ⁴⁴¹VFNN⁴⁴⁴ or ⁴⁴⁸VQIG⁴⁵¹ in RIPK3 led to the complete disruption of the TNF-induced cell necroptosis. The NIH-3T3 cells infected with lentivirus containing FKBPv fused wild-type or mutant RIPK3 were treated with TNF-α/Smac/z-VAD (T/S/Z) 10 h. The number of surviving cells were determined by measuring ATP levels using Cell Titer-Glo kit (upper). Data are presented as mean ± SD of n = 3 biologically independent replicates. Source data are provided as a Source Data file. Aliquots of 20 μg whole-cell lysates were subjected to SDS-PAGE followed by western-blot analysis of mouse RIPK3 and β-Actin which was shown as a loading control (lower). c The RIPK3 mutant F442D, Q449D, L456D did not affect the interaction between mouse RIPK1 and mouse RIPK3. The HEK293T cells were co-transfected with DNA plasmids containing mouse RIPK1 and Flag-tagged mouse RIPK3 (or its mutants). Cell lysates were collected 36 h post transfection, and immunoprecipitated with anti-Flag magnetic beads (Bimake) at 4 °C. The total cell lysates and immunoprecipitates were analyzed by western-blot analysis with the indicated antibodies. All experiments were repeated three times. The following antibodies were used in this study: anti-mRIPK3 (Sigma-Aldrich, PRS2283, 1:3000); anti-RIPK1 (Cell Signaling Technology, D94C12, 1:1000); Anti-actin (MBL, PM053-7, 1:10,000). Uncropped blots in the Source Data file.

Fig. 8. Molecular dynamics (MD) simulations using Xplor-NIH on mouse RIPK3 fibrils and a hetero-amyloid model of mouse RIPK1/RIPK3.

a The best four structures of mouse RIPK3 fibril after 50 ps MD, showing the fibril developing a left-hand twist. b The structure alignment of RIPK3 conserved tetrad sequence from human RIPK1/RIPK3 hetero-amyloid structure (purple, 5v7z.pdb), mouse RIPK3 fibril structure (blue, 6JPD.pdb) and mouse RIPK3 fibril after 50 ps MD from (a) (cyan). c The best four structures of mouse RIPK1/RIPK3 hetero-amyloid after 50 ps MD run, showing the opening the β-arches formed by the 1st and 2nd β-strand. The mouse RIPK3 fibril structure was adopted as the starting configuration for the MD. The sequence alignment for mouse RIPK1 and RIPK3 is shown on the top. d The structure comparison between c (cyan, showing only the best two structures for clarity, residue V448, I450 was also shown in one subunit) and RIPK1/RIPK3 from the human RIPK1/RIPK3 hetero-amyloid structure (purple, 5v7z.pdb). e The proposed mechanism showing RIPK3 structural transformation from initial RIPK1–RIPK3 binding to RIPK3 fibril formation.