1. Structural Insights into RIP3-Mediated Necroptotic Signaling
Tian Xie, Wei Peng, Chuangye Yan, Jianping Wu, Xinqi Gong, Yigong Shi 
Cell Reports 
Volume 5, Issue 1, Pages (October 2013)
DOI: /j.celrep
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2. Cell Reports 2013 5, 70-78DOI: (10.1016/j.celrep.2013.08.044)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Overall Structure of the Mouse RIP3 Kinase Domain
(A) Overall structure of the mouse RIP3 kinase domain (colored green). The P-loop (residues 29–36) and the αC helix (residues 57–66) are colored pink and purple, respectively. The activation loop (residues 161–194) is highlighted in red; residues 171–185 are disordered in the crystals. All structural figures were prepared with PyMOL (DeLano, 2002).
(B) Lys51 forms a H-bond with Glu61 from the αC helix of RIP3. The side chains of Lys51 and Glu61 are shown in sticks. H-bonds in this and all other figures are represented by red dashed lines.
(C) H-bond between Phe162 and Ser165 of RIP3 helps to stabilize the DFG motif in the active configuration.
(D) The hydrophobic spine structure in RIP3. The side chains of the four amino acids, shown in sticks, stack against each other in a linear fashion.
(E) Crystal lattice packing interactions between two RIP3 molecules in an asymmetric unit. The side chain of Arg69 in one molecule (Mol B) makes two H-bonds to the main chain groups of Val66 and Leu76 in the other molecule (Mol A), and vice versa.
See also Figure S1.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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4. Figure 2 In Vitro Binding and Phosphorylation of MLKL by RIP3
(A) Analysis of the interactions between mouse RIP3 kinase domain (RIP3-KD) and mouse MLKL kinase-like domain (MLKL-KL) by gel filtration. The same elution fractions of each gel filtration run were applied to SDS-PAGE followed by Coomassie blue staining. Individually expressed and purified RIP3 and MLKL failed to form a stable complex, whereas coexpressed RIP3 and MLKL formed a stable complex.
(B) In vitro phosphorylation of wild-type and mutant MLKL by RIP3. MLKL was phosphorylated at Ser345, Ser347, Thr349, and Ser352. Mutation of any of these four residues to Ala or Glu still allowed phosphorylation by RIP3, whereas the quadruple mutants S345A/S347A/T349A/S352A (ST4A) and S345E/S347E/T349E/S352E (ST4E) failed to be phosphorylated.
(C) Overall structure of mouse MLKL kinase-like domain (colored cyan). The four phosphorylation sites by RIP3 are located at an intervening region between the N- and C-lobes of MLKL. Ser345 and Ser347 are colored red, and Thr349 and Ser352 are disordered and invisible.
See also Figure S2.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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5. Figure 3
Structure of the Mouse RIP3-MLKL Complex and the Interface between RIP3 and MLKL
(A) Overall structure of the mouse RIP3-MLKL complex. The RIP3 kinase domain and MLKL kinase-like domain are colored green and cyan, respectively. AMP-PNP is shown in magenta ball and stick.
(B) The interface between the N- and C-lobes of RIP3 and MLKL. The interface residues are shown in sticks.
(C) A close-up view of the N-lobe interface between RIP3 and MLKL.
(D) A close-up view of the H-bond network in the C-lobe interface between RIP3 and MLKL.
(E) A close-up view of the hydrophobic interactions in the C-lobe interface between RIP3 and MLKL. The right panel shows the hydrophobic cavity surrounding Phe373 from MLKL.
See also Figure S3.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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6. Figure 4
Conformational Changes of RIP3 and MLKL upon RIP3-MLKL Complex Formation
(A) Structural comparison of free RIP3 and MLKL-bound RIP3. Free RIP3 is colored yellow, with its αC helix in magenta and T-loop in orange. MLKL-bound RIP3 is colored green, with its αC helix in purple and T-loop in red. AMP-PNP is shown in magenta sticks.
(B) A close-up view of the conformational change in the N-lobe of RIP3 in response to MLKL binding. The side chains of Ser228 from MLKL and Lys56 and Ser89 from RIP3 are shown in sticks.
(C) The movement of αC helix leads to disruption of the salt bridge between Lys51 and Glu61 in RIP3.
(D) A close-up view of the conformational change in the DFG motif of RIP3 in response to MLKL binding.
(E) A close-up view of the rearrangement of the hydrophobic spine structure in RIP3 in response to MLKL binding.
(F) Structural comparison of free MLKL and RIP3-bound MLKL. Free MLKL is colored orange, with its α1 and α4 helices in pink. RIP3-bound MLKL is colored cyan, with its α1 and α4 helices in yellow. RIP3 is represented in green surface. AMP-PNP is shown in magenta sticks.
(G) A close-up view of the conformational changes of the residues in the N-lobe of MLKL in response to RIP3 binding. The residues are shown in sticks.
See also Figure S4.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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7. Figure 5 Homology Modeling of the Human RIP3-MLKL Complex
(A) A structural model of the human RIP3 kinase domain.
(B) The crystal structure of human MLKL kinase-like domain.
(C) A structural model of the human RIP3-MLKL complex. The human RIP3 and MLKL are colored dark green and light cyan, respectively.
(D) Structural comparison of the mouse RIP3-MLKL complex and the human RIP3-MLKL complex. The mouse RIP3 and MLKL are colored green and cyan, respectively. The human RIP3 and MLKL are colored dark green and light cyan, respectively.
(E) Phe386 in human MLKL inserts into a hydrophobic pocket in the C-lobe of human RIP3 on the basis of the modeled human RIP3-MLKL structure. The surface of human RIP3 is represented by electrostatic potential. Phe386 is shown in sticks.
See also Figure S5.
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8. Figure S1
Structural Features of the Mouse RIP3 Kinase Domain, Related to Figure 1
(A) Sequence alignment of the kinase domains of four RIP3 orthologs. mRIP3: Mus musculus RIP3; hRIP3: Homo sapiens RIP3; xRIP3: Xenopus laevis RIP3; dRIP3: Danio rerio RIP3. Conserved residues are highlighted in yellow. Secondary structural elements of RIP3 are indicated above the sequences, with the T-loop α-helix colored red. The P-loop, catalytic loop, and the DFG and APE motifs in T-loop are highlighted in magenta. The catalytic triad residues Lys51/Glu61/Asp161 are indicated with red triangles. The N-lobe residues mediating interactions between RIP3 and MLKL are identified by blue circles. The C-lobe residues mediating the H-bond network between RIP3 and MLKL are indicated with green diamonds. The residues surrounding the hydrophobic pocket for Phe373 in MLKL are identified by magenta stars.
(B) Structural comparison of RIP3 and PKA (PDB code 2CPK). PKA is colored light gray. RIP3 is colored green, with its P-loop in pink, αC helix in purple and T-loop in red. The side chains of Lys72 and Glu91 of PKA and Lys51 and Glu61 of RIP3 are shown in sticks.
(C) A network of conserved H-bonds around the DFG motif in PKA of the active configuration.
(D) The stacked spine structure in PKA. The side chains of the spine structure are shown in sticks.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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9. Figure S2
In Vitro Binding of MLKL by RIP3 and Structural Features of Mouse MLKL Kinase-like Domain, Related to Figure 2
(A) Identification of the phosphorylation sites in the mouse MLKL kinase-like domain. The tandem mass (MS/MS) spectrum of a duple-charged ion at m/z for MH22+ corresponding to the mass of the quadruple-phosphorylated peptide TQNSISRTAKSTK. The labeled peaks corresponding to masses of b and y type product ions of the peptide suggested this quadruple-phosphorylated peptide to be TQNpSIpSRpTAKpSTK, with Ser345, Ser347, Thr349, and Ser352 as the phosphorylated amino acid residues. (B) Analysis of the interactions between mouse RIP3 kinase domain (RIP3-KD) and mouse MLKL kinase-like domain quadruple mutant ST4A (S345A/S347A/T349A/S352A) by gel filtration. The same elution fractions of each gel filtration run were applied to SDS-PAGE followed by coomassie blue staining. Individually expressed and purified RIP3 and MLKL failed to form a stable complex, whereas coexpressed RIP3 and MLKL formed a stable complex.
(C) Analysis of the interactions between mouse RIP3 kinase domain (RIP3-KD) and mouse MLKL kinase-like domain quadruple mutant ST4E (S345E/S347E/T349E/S352E) by gel filtration.
(D) Analysis of the interactions between further phosphorylated mouse RIP3 kinase domain (RIP3-KD (P)) and mouse MLKL kinase-like domain by gel filtration. Further phosphorylated RIP3 was able to form a stable complex with MLKL.
(E) Sequence alignment of the kinase-like domains of mouse MLKL (mMLKL) and human MLKL (hMLKL). Conserved residues are highlighted in yellow. Secondary structural elements of MLKL are indicated above the sequences. The N-lobe residues mediating the binding between RIP3 and MLKL are indicated with blue circles. The C-lobe residues mediating the H-bond network between RIP3 and MLKL are indicated with green diamonds. The Phe373 which inserts into the hydrophobic pocket in RIP3 is indicated with magenta stars. The phosphorylation sites in MLKL by RIP3 are indicated with red triangles.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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10. Figure S3
Structural Features of the Mouse RIP3-MLKL Complex and Assessment of Interactions between Variants of the Mouse RIP3 Kinase Domain and the Mouse MLKL Kinase-like Domain, Related to Figure 3
(A) Overall structure of the mouse RIP3-MLKL complex in surface representation. RIP3 is colored green. MLKL is colored cyan. AMP-PNP is shown in magenta sticks.
(B) A close-up view of the 2Fo-Fc electron density map, contoured at 2.5σ, is shown for AMP-PNP in the mouse RIP3-MLKL complex.
(C) Mutation of Phe234 in MLKL or Phe27 in RIP3 to Glu crippled formation of the RIP3-MLKL complex. For each experiment (each lane), the two proteins were coexpressed in Sf-9 cells and purified by Ni-affinity resin. The MLKL kinase-like domain contains an N-terminal 10xHis tag.
(D) Mutation of Phe373 to Ala or Arg in MLKL led to complete disruption of the RIP3-MLKL complex formation.
(E) Assessment of impact of mutating the residues that are involved in H-bonds at the C-lobe interface. Whereas mutation of the phosphorylated residue Ser232 is deleterious for formation of the RIP3-MLKL complex, mutation of other residues had little impact.
(F) The quadruple mutations of the phosphorylation sites in MLKL (ST4A: S345A/S347A/T349A/S352A, and ST4E: S345E/S347E/T349E/S352E) failed to disrupt formation of the RIP3-MLKL complex.
(G) Mutation of Thr231 in RIP3 failed to disrupt formation of the RIP3-MLKL complex.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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11. Figure S4
Structural Comparison of MLKL-Bound RIP3 and Bisubstrate-Analog-Bound ABL1, Related to Figure 4
(A) A close-up view on the H-bonds from MLKL-bound RIP3 residues to the magnesium ion coordinated AMP-PNP. The P-loop and catalytic loop in RIP3 are colored pink and yellow, respectively. The residues contributing to H-bond formation with AMP-PNP and magnesium ion coordination are shown in sticks. AMP-PNP is shown in magenta ball-and-stick. Magnesium ion is represented by gray sphere.
(B) A close-up view on the van der Waals contacts to the adenine base and ribose of AMP-PNP. The hydrophobic amino acids surrounding adenosine in RIP3 are shown in sticks.
(C) Structural comparison of MLKL-bound RIP3 and bisubstrate-analog-bound ABL1. MLKL-bound RIP3 is colored green. Bisubstrate-analog-bound ABL1 is colored light orange. AMP-PNP is shown in magenta sticks. ABL1 bisubstrate analog is shown in light gray sticks.
(D) A close-up view of the nucleotide-binding site in MLKL-bound RIP3 and bisubstrate-analog-bound ABL1. The catalytic triad residues Lys51/Glu61/Asp161 and catalytic-loop residue Asn148 in MLKL-bound RIP3 and the corresponding Lys271/Glu286/Asp381 and Asn368 residues in bisubstrate-analog-bound ABL1 are shown in sticks. The magnesium ions in MLKL-bound RIP3 and bisubstrate-analog-bound ABL1 are represented by gray and yellow spheres, respectively. The water molecules in MLKL-bound RIP3 and bisubstrate-analog-bound ABL1 are represented by blue and pink spheres, respectively.
(E) A close-up view of the magnesium ion coordinated by the α-, β-, and γ-phosphates of AMP-PNP, Asp161, Asn148, and a water molecule in MLKL-bound RIP3 (left panel); a close-up view of the magnesium ion coordinated by the α- and γ-phosphates of ATP analog, Asp381, Asn368, and two water molecules in bisubstrate-analog-bound ABL1 (right panel).
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
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12. Figure S5
Interface between RIP3 and MLKL in Human RIP3-MLKL Complex Model, Related to Figure 5
(A) A close-up view of the H-bond network in the C-lobe interface between RIP3 and MLKL in the human RIP3-MLKL complex model. The human RIP3 and MLKL are colored dark green and light cyan, respectively. The residues contributing to H-bond formation are shown in sticks.
(B) A close-up view of the N-lobe interface between RIP3 and MLKL in the human RIP3-MLKL complex model.
Cell Reports 2013 5, 70-78DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions