1. Volume 25, Issue 1, Pages 66-78 (January 2017)
Structural Insights into Linear Tri-ubiquitin Recognition by A20-Binding Inhibitor of NF- κB, ABIN-2 
Shan-Meng Lin, Su-Chang Lin, Jhen-Yi Hong, Tsung-Wei Su, Bai-Jiun Kuo, Wei-Hsin Chang, Yi-Fan Tu, Yu-Chih Lo 
Structure 
Volume 25, Issue 1, Pages (January 2017)
DOI: /j.str
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2. Structure 2017 25, 66-78DOI: (10.1016/j.str.2016.11.005)
Copyright © 2016 Elsevier Ltd Terms and Conditions

3. Figure 1 The Structure of the hABIN2:triUb Complex
(A) Domain organization of hABIN2 and hNEMO. The fragment of ABIN2 used for the reconstitution of the hABIN2:triUb complex is shown in the red box. AHD, ABIN homology domain; HLH, helix-loop-helix; CC, coiled-coil; LZ, leucine-rich region; ZF, zinc finger region.
(B) The gel-filtration profile of the complex and the corresponding SDS-PAGE of the peak fractions. The complex was eluted in fractions 12 and 13. M, molecular weight marker. B, BSA; C, ABIN2 alone; In, the sample before the injection to fast protein liquid chromatography. Fractions 12 and 14 were labeled with a blue dot.
(C) The 2Fo − Fc electron density map of the contact region between hABIN2 and triUb, as shown in the gray box in (D), shows that some hABIN2 residues (labeled in black) recognize the M1-linkage by interacting with R74 of the 1stUb (labeled in blue) and Q2, F4 of the 2ndUb (labeled in pink). The map is contoured at 1.5σ by using the program Coot.
(D) Cartoon representation of the crystal structure of the hABIN2:triUb complex. The pink lines show the helical axes of the main helices that interact with triUb.
(E) Same as (D), but rotated 60° along the y axis. The orange lines indicate the approximate N-to-C direction of the ABIN2 dimer and the 1stUb-2ndUb fragment, respectively. Because the β sheet of Ub is curved, we used a line that passes through the Cα atoms of both H68 in the 1stUb-2ndUb fragment to represent its N-to-C direction.
Structure  , 66-78DOI: ( /j.str )
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4. Figure 2 Sequence and Structural Comparisons of ABIN2 and NEMO
(A) The structure-based sequence alignment of hABIN2 and NEMO. The “a” and “d” positions of hABIN2 and NEMO are shown in black and blue, respectively. The continuous nine-heptad repeats of hABIN2 are numbered from 1 to 9 and separated by blue lines. The predicted “a” and “d” positions of the unstructured hABIN2 sequence are shown in gray. The MET stammer and cysteine residues are highlighted in a red box and in blue, respectively. Human disease mutations of NEMO are shaded in yellow. The main different Ub-interacting residues between ABINs and NEMOs are highlighted in purple. hABIN2 residues for point-mutagenesis are underlined with the substituents shown above the alignment. The mutations that disrupt Ub-binding are shown in red. The ABIN1, ABIN3, and OPTN sequences were aligned to the ABIN2 sequence. The inserted sequences of “IQTPPSSP” and “IKC” of ABIN1 and ABIN3 were deleted, respectively, for a better alignment for AHD1, and the green line shows the deletion site.
(B) Local coiled-coil parameters of the hABIN2 structure plotted against the residue number. cc_rad, coiled-coil radius (Å, green line); cc_I_pit/100, coiled-coil pitch/100 (Å, red line); cc_dang, coiled-coil phase (degree, purple line); a_dang-100, coiled-coil-100 (degree, cyan line); res/turn, the periodicity of the coiled-coil (pink line). The periodicity (residue/turn) series was plotted against the secondary axis.
(C) Same as (B) but using the mNEMO structure. The stammer is highlighted in pink.
(D) The polar amino acids at the “a” or “d” position of hABIN2 are shown as spheres and labeled in black.
(E) Structural comparison of hABIN2 and mNEMO (PDB: 2ZVN) by superimposing the lower left helices of the primary Ub-binding sites. mNEMO P292 is shown as a sphere. The MET stammer is colored and labeled in red.
(F) Same as (E) but is rotated by 90° along the x axis, to show that hABIN2 and NEMO have different bending angles of the coiled-coil axis.
(G) Structural comparison of the hABIN2 dimer by superimposing the lower left helix of the secondary Ub-binding site to that of the primary Ub-binding site to show that hABIN2 has a curved coiled-coil axis and also to show the difference in Ub-binding between the primary (shown with colors) and secondary (shown in gray) Ub-binding sites. Gray arrows point out the model is bending toward a different direction.
Structure  , 66-78DOI: ( /j.str )
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5. Figure 3 The Interactions between hABIN2 and TriUb
(A) The crystal packing of the hABIN2:triUb complex, in which triUb simultaneously interacts with two hABIN2 in a similar way. The 1stUb-2ndUb region binds the primary Ub-binding site of hABIN2 and the 2ndUb-3rdUb binds the secondary Ub-binding site of another hABIN2. The black spot at the center of the colored triUb is the approximate intersecting location of the helical axis of the triUb, which is perpendicular to the paper.
(B) The left panel shows the interaction residues between hABIN2 and Ub on the primary Ub-binding, which is divided into the “Left” and “Right” sites with their Ub-interacting residues labeled in red and blue, respectively. The ABIN2-interacting residues of the 1stUb and 2ndUb are shown as slate and pink sticks, respectively. The right panel shows the corresponding surface representation of the primary Ub-binding site. The critical hABIN2-interacting residues on the 1stUb and 2ndUb are labeled in blue and red, respectively. The five hot spots for hABIN2:triUb interactions are labeled with white circles. Detail interactions in the black and the green dashed boxes are shown in (C) and (D), respectively. The electrostatic potential surface of hABIN2 was plotted at ± 5 kT/e (red, negative; blue, positive).
(C) The interaction between hABIN2 and 1stUb. Two major interaction regions are labeled with yellow dashed circles. The hot spots for hABIN2:triUb interactions are labeled with white circles.
(D) The interaction between hABIN2 and 2ndUb. Two major interaction regions are labeled with yellow dashed circles. The hot spots for hABIN2:triUb interactions are labeled with white circles.
See also Figures S1 and S2 and Tables S1 and S2.
Structure  , 66-78DOI: ( /j.str )
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6. Figure 4 Studying the hABIN2:Ub Interaction in Solution
(A) ITC analysis of the interaction between hABIN2 and triUb. Concentrated triUb was titrated into a hABIN2 solution.
(B) Molecular weight analysis of the hABIN2:triUb complex by gel filtration coupled with multi-angle light scattering (SEC-MALS). The sold line is the absorption of UV. The dashed line is the apparent molecular weight of the complex in the peak fractions. The SDS-PAGE of each peak fraction is shown below. The width of each protein band represents the width of the corresponding peak fraction.
(C) ITC analysis of the interaction between hABIN2 and linear diUb by titrating diUb into hABIN2.
(D) ITC analysis of the interaction between hABIN2 and the triUb 2nd-3rd L73A double mutant by titrating the triUb mutant into hABIN2.
Structure  , 66-78DOI: ( /j.str )
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7. Figure 5
Comparisons of the Ub Units of TriUb and Their Footprints on hABIN2
(A) Surface representation of the primary Ub-binding site of hABIN2 with the residues colored according to the size of the buried surface (BSA) shown in Table S1. Cyan, BSA >70 Å2; red, 70–45 Å2; pink, 45–20 Å2.
(B) Same as (A) but showing the secondary Ub-binding site.
(C) Superimposition of the 1stUb with the 2ndUb of triUb with a root-mean-square deviation (RMSD) of 0.262 Å.
(D) Superimposition of the 2ndUb with the 3rdUb of triUb with an RMSD of 0.505 Å.
(E) Superimposition of the 1stUb with the 3rdUb of triUb with an RMSD of 0.384 Å.
See also Figures S1 and S2 and Tables S1 and S2.
Structure  , 66-78DOI: ( /j.str )
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8. Figure 6 The hABIN2-Interacting Residues of TriUb
(A) TriUb on the primary Ub-binding site as viewed from the ABIN2 (shown as a transparent electrostatic surface). The quadrilateral that represents the curved β sheet of Ub is shown as blue, magenta, and green dotted lines on the 1stUb, 2ndUb, and 3rdUb, respectively. The four residues of Q2, G10 and G47, and L71 that represent the four vertices of the quadrilateral are shown as sticks, respectively, on each Ub. The residues critical for the recognition by hABIN2, which were determined by the pull-down assays with the triUb triple mutants in Figure 7C, are shown as red sticks and labeled with the residue name in a red box.
(B) Another view of triUb made by rotating the model in (A) by 30° along the x axis. The 1stUb and 2ndUb show, approximately, the front and side views of the quadrilateral, respectively. The angle between the two blue lines passing through the Cα atoms of Q2 and H68 and the Cα atoms of H68 and L71, respectively, is 119° and labeled as α. G76 and K63 are also shown as sticks. The distance between the NZ atom of K63 and the oxygen atom of G76 is 3.8 Å.
(C) The residues on the F4, I36, and I44 patches and the TEK box are shown as cyan, green, yellow, and orange sticks, and are labeled individually on the 1stUb. The location of each patch and TEK box are shown using dashed circles with the color corresponding to the sticks. The orientation of this model is the same as that in (B). On the 2ndUb, the residues of each patch and TEK box are also shown as sticks.
(D) The triangular pyramid represents the approximate 3D shape of the curved β sheet of the 2ndUb shown in (B) and in the same orientation. The shaded surface is mainly for the hABIN2 interaction.
(E) The electrostatic potential surface of triUb in (A) plotted at ± 5 kT/e (red, negative; blue, positive). The approximate locations of critical residues for the hABIN2:triUb interaction are shown in a red dashed box.
(F) The electrostatic potential surface of triUb in (B). The approximate locations of critical residues for the hABIN2:triUb interaction are shown in a red dashed box.
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9. Figure 7
The Pull-Down Assays on the hABIN2:triUb and hABIN2:diUb Interactions
(A) The upper part shows the SDS-PAGE showing the triUb pulled down by the wild-type (WT) hABIN2 and also various hABIN2 mutants. The lower part shows the unbound fraction in each experiment. Each SDS-PAGE has a lane for WT hABIN2 as a positive control. Control, resin mixed with triUb alone as a negative control. The pull-down intensity is shown in parentheses.
(B) The diUb pull-down assays to show the mutational effect on the hABIN2:diUb interaction for comparison with the result in (C). The mutation created on the first or second Ub is labeled with the mutation followed by 1st or 2nd, respectively. A mutant with mutations on both Ubs is labeled with “double.” Control, resin mixed with diUb alone as a negative control.
(C) The triUb pull-down assays to find the triUb residues critical for hABIN2:triUB interaction. The mutation is created on either the first, second, or third Ub, which is labeled with the mutation followed by 1st, 2nd, or 3rd, respectively. Triple mutation of some specific residue on triUb is labeled with the mutation followed by “triple.” The L73A double mutation on the 1stUb and 2ndUb is labeled as L73A-1st-2nd, while the L73A double mutation on the 2ndUb and 3rdUb is labeled as L73A-2nd-3rd. The unbound fractions show that the input amount of each triUb protein is about the same.
Structure  , 66-78DOI: ( /j.str )
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