1. Volume 20, Issue 10, Pages 1704-1714 (October 2012)
The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site 
Gladys de Leon-Boenig, Krista K. Bowman, Jianwen A. Feng, Terry Crawford, Christine Everett, Yvonne Franke, Angela Oh, Mark Stanley, Steven T. Staben, Melissa A. Starovasnik, Heidi J.A. Wallweber, Jiansheng Wu, Lawren C. Wu, Adam R. Johnson, Sarah G. Hymowitz 
Structure 
Volume 20, Issue 10, Pages (October 2012)
DOI: /j.str
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2. Structure 2012 20, 1704-1714DOI: (10.1016/j.str.2012.07.013)
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3. Figure 1 Domain Structure of NIK Kinase
(A) Schematic diagram of human NIK. Human and murine NIK numbering differs by two amino acids. We indicate murine and human numbering with a superscript “m” and “h,” respectively. The N-terminal region of NIK includes the TRAF3 binding site (residues 78h–84h, shown as a gray oval) and the nuclear localization signal (residues 136h–143h, shown as a lavender oval). The core kinase domain is shown as a light blue rectangle flanked by red and blue rectangles indicating N- and C-terminal sequences (327h–375h and 655h–675 h, respectively) that are conserved in NIK from multiple species, but divergent from other Serine/Threonine kinases.
(B) Alignment of murine and human NIK kinase domains to the core kinase region of PKA (residues 40–293). The conserved additions to the NIK kinase domain are colored as in (A). The P loop is orange and the V408m/L406h difference is underlined. The gatekeeper residue 469h (underlined) and the hinge region is green. Conserved kinase features are highlighted in lavender, while T559h is shown in light blue. The phosphothreonine in PKA (Thr 197) is shown in bold. Secondary structure elements present in Apo mNIK are shown as lavender cylinders (helices) and green arrows (beta strands). Additional information on expression constructs is found in Table S1.
Structure  , DOI: ( /j.str )
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4. Figure 2 Structure of Apo NIK Kinase Domain
(A) Structure of murine NIK kinase colored as in Figure 1B.
(B) Close-up of the hydrophobic interactions made by the residues 345m–363m with the αC helix.
(C) Final 2Fo-Fc (blue, 1σ) and Fo-Fc (green, 4σ) electron density map shows the nucleotide-binding site is unoccupied except for the side chain of Arg 410m.
(D) Superposition of the active-like conformation of PKA (taupe, PDB code 1ATP) and apo mNIK colored as in (A) showing the apo mNIK conformation has features of an active kinase. Residues of interest are shown as sticks including the salt bridge formed in PKA between the phosphothreonine Thr 197 and the Arg 165 from the HRD motif. The Cα atom of Phe of the GxGxΦ motif from both the structures is shown with a sphere as the Cα atom of Gly 516m.
(E) Structure of human NIK kinase at 2.9 Å (green) superimposed on mNIK kinase colored as in (A) showing the similarity in the mNIK and hNIK structures. Additional information on crystal packing is included in Figure S1.
Structure  , DOI: ( /j.str )
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5. Figure 3
Unphosphorylated NIK Kinase Domain Is Sufficient for ATPase and Kinase Activity
(A) Plot of initial velocity versus ATP concentration for mNIK 329m–667m kinase domain in an assay monitoring intrinsic ATPase activity. This assay used 100 nM of NIK (wild-type proteins) or 600–700 nM NIK (mutant proteins). The Km for ATP and Vmax calculated for mNIK from this plot, as well as the kinetic constants for additional NIK proteins, are summarized in Table 2. Vmax was normalized to 100 nM enzyme concentration.
(B) Kinase activity of NIK. The plot shows the phosphorylation of a peptide containing the Ser 176 phosphorylation site in IKKα versus NIK concentration for the human NIK 327h–673h kinase domain (solid line), a phosphorylated hNIK 327h–673h kinase domain (dotted line), and a hNIK protein (318h–947h) that includes the entire C terminus (dashed line). The error bars represent the standard deviation of multiple measurements. All three NIK proteins show largely equivalent ability to phosphorylate this peptide fragment of IKKα. Supporting assay data are included in the Supplemental Experimental Procedures and Figure S2.
Structure  , DOI: ( /j.str )
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6. Figure 4
Structure of mNIK Bound to Diverse Inhibitors Reveals Conformational Plasticity
(A) Cα ribbon image of mNIK (colored as in Figure 2A) covered by a transparent surface bound to cmp1 (carbon atoms colored magenta) showing cmp1 is largely sequestered from solvent. The P loop (orange) retains the conformation seen in the structure of apo mNIK.
(B) Details of interactions cmp1 (carbon atoms colored pink) makes with mNIK (carbon atoms colored yellow with a transparent white surface). Potential hydrogen bonds are shown as dashes. Water molecules are shown as small spheres.
(C) Superposition of mNIK with cmp1 (carbon atoms colored yellow) on mNIK bound to cmp2 (carbon atoms colored dark green) showing the backbone conformations are very similar except for the P loop. Cmp2 is shown as spheres with carbon atoms colored violet.
(D) Details of the reconfiguration of the P loop and the gatekeeper residue M417m are shown in the superposition of the structures of mNIK bound to cmp1 (yellow) and mNIK bound to cmp2 (green). Additional information is included in Figure S3.
(E) Close-up of the interactions made by cmp1 with mNIK. The mNIK protein is shown as green sticks and a transparent white surface. Hydrogen bonds are shown as dashed lines.
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7. Figure 5 The V408mL Mutation “Humanizes” the Murine NIK Kinase Domain
(A) Structure of mNIK 345m–675m V408mL bound to cmp3.
(B) Superposition of mNIK 345m–675m V408mL (blue) on mNIK 345m–675m (yellow and white transparent surface) showing the presence of a valine in the mNIK makes the pocket slightly smaller due to the position of the valine methyl group.
(C) Plot of the log(Ki) for hNIK (327h–673h) and mNIK (329m–675m) for 32 compounds tested in triplicate titrations in an ATPase activity assay, including staurosporine (red square). The data are fit with a line with R2 = 0.83, Slope = 1.01 and an intercept of The Ki of staurosporine for hNIK is 15 nM and for mNIK is 83 nM based on two to four measurements.
(D) Comparison of Ki values for hNIK (327h–673h) and mNIK (345m–675m) V408L for the same 32 compounds. The data are fit with a line with R2 = 0.94, slope = 1.03, and an intercept of − The Ki of staurosporine (red square) for mNIK (345m–675m) V408mL is 4.3 nM.
See also Figure S4.
Structure  , DOI: ( /j.str )
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