Volume 25, Issue 6, Pages 867-877.e3 (June 2017) Structural Basis of TPR-Mediated Oligomerization and Activation of Oncogenic Fusion Kinases  Kuntal Pal, Abhishek Bandyopadhyay, X. Edward Zhou, Qingping Xu, David P. Marciano, Joseph S. Brunzelle, Smitha Yerrum, Patrick R. Griffin, George Vande Woude, Karsten Melcher, H. Eric Xu  Structure  Volume 25, Issue 6, Pages 867-877.e3 (June 2017) DOI: 10.1016/j.str.2017.04.015 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Schematic Representation of the TPR-MET Chromosomal Translocation (A) Structural and functional domains of MET receptor, translocated promoter region (TPR), and the TPR(1–142)-MET(1,010–1,390) fusion protein. Chromosomal translocation mediated fusion between the TPR and the MET receptor genes are indicated by blue lines. Translocation results in expression of a cytosolic, constitutively active TPR-MET oncogene. (B) MET consists of a large extracellular region that contains a Sema domain, a cysteine-rich (CR) domain, and four immunoglobulin (IG) domains followed by a single transmembrane helix and the intracellular juxtamembrane (JMD) and kinase domains (KD). Hepatocyte growth factor (HGF)-regulated MET dimerization in the extracellular region transmits a signal for kinase domain autophosphorylation and downstream signaling. TPR-MET lacks the MET regulatory extracellular domain and forms a dimeric, constitutively active fusion kinase oncogene. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Structural Organization of Dimeric TPR (A) Crystal structure of TPR(2–142). TPR forms a parallel coiled-coil dimer with chain A colored in green and chain B in cyan that further oligomerizes to form antiparallel tetramers (red and yellow chains). (B) Ribbon diagram of the helical caps of chain A (green) and chain B (cyan) with hydrophobic and ionic interactions indicated by dashed lines. (C–F) Stick representations of dimerization interface residues in the N-terminal coiled coil (C), the first leucine zipper (residues 77–100, D), the second leucine zipper (residues 120–141, E), and the middle region with a C75-C75 disulfide bond found in one of the tetramers in the asymmetric unit (F). The side chains of “a” and “d” residues in heptad (a-b-c-d-e-f-g) repeats of leucine zippers are displayed. (G) Disulfide crosslinking validation of the parallel arrangement of the TPR dimer. Cells transfected with TPR mutant constructs were treated with H2O2 and lysed, lysates separated by non-reducing SDS-PAGE, and TPR and TPR crosslinking adducts detected by immunoblotting. See also Figures S1 and S2. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Tetrameric Organization of TPR (A) TPR(2–142) tetramer in ribbon presentation, in which one dimer is overlaid with a transparent surface electrostatic potential map. Blue-colored regions indicate positive charge and red-colored regions negative charge (see charge potential heatmap bar below). The four monomers are colored green, cyan, magenta, and yellow. (B) Tetramer distribution of TPR(2–142) wild-type (WT) and TPR-F55M/F113M(2–142) determined by size-exclusion chromatography. (C) Validation of the antiparallel tetramer arrangement by in cell disulfide crosslinking between indicated residues of chain A and chain C lysates of H2O2-treated FLAG-TPR-MET-expressing cells were analyzed by non-reducing SDS-PAGE and immunoblotting with anti-FLAG antibody. (D) Parallel tetramer orientation of TPR(2–142) F113M. (E) Dimeric arrangement of TPR(2–142) F55M/F113M. See also Figure S3. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 The TPR Dimer-Dimer Interface Is Mediated Largely by Ionic Interactions (A) Cartoon structure of the dimer-dimer interface between chain A (purple) and chain C (magenta). Key interface residues are shown in stick representation. (B) Size column profiles of TPR-MET wild-type (WT) and TPR mutant proteins with charge reversal mutations of tetramer interface residues. Elution of size standards is indicated by arrows. The molecular weight of the monomeric TPR-MET fusion protein is 55.6 kDa, indicating that the peaks correspond to aggregated (∼122 mL elution volume) and predominantly tetrameric (∼169 mL) TPR-MET. (C) Immunoblot of lysates from AD293 cells expressing TPR-MET wild-type and tetramer interface mutants. TPR activity of mutant proteins was probed with antibodies that recognize the TPR kinase domain phosphorylated at Y1234 and Y1235 (pMET). Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 TPR-MET Forms a Stable Tetramer Size-exclusion chromatogram of TPR-MET at different concentrations. Blue line: chromatogram of TPR-MET Ni eluate, which resolved two major peaks and allowed us to isolate the predominantly tetramer peak at ∼169 mL elution volume and a concentration of 520 nM protein. We confirmed tetramer formation in the 169-mL eluate by MALS (calculated molecular weight, 193 kDa), and reloaded at lower concentrations onto the size-exclusion column, resulting in only slightly right-shifted peaks at concentrations of 52 nM (brown line) and 26 nM (red line), indicating stable tetramer formation. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Hydrogen Deuterium Exchange Mass Spectrometry Analysis of Monomeric and Tetrameric MET Kinase Domains (A) MET kinase HDX perturbation map. Heatmap of the difference in hydrogen deuterium exchange between the monomeric MET kinase domain and the tetrameric domain in the context of TPR(2–142)-MET overlaid on the MET kinase domain structure (PDB: 3Q6U). (B) Structure overlay of MET kinase domain in active (PDB: 3Q6U, green), inhibitor-bound inactive (PDB: 2WD1, orange), and non-phosphorylated inactive (PDB: 2G15, gray) conformations. See also Figure S4. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Mutational Analysis of the TPR Leucine Zipper and the MET Juxtamembrane Domain (A) Leucine residues of the two TPR leucine zippers were mutated individually or in combination. Wild-type (WT) and mutant total (MET) and active, phosphorylated (pMET) TPR-MET were detected by immunoblotting. (B) Autophosphorylation of TPR-MET variants with truncations in the MET juxtamembrane domain. Left: schematic of constructs analyzed. Right: immunoblot probed with antibodies for total (MET) and active, phosphorylated (pMET) TPR-MET. See also Figure S5. Structure 2017 25, 867-877.e3DOI: (10.1016/j.str.2017.04.015) Copyright © 2017 Elsevier Ltd Terms and Conditions