ITC data for CDC37-BRAF interactions for paper: Recognition of BRAF by CDC37 and Re-evaluation of the Activation mechanism for the Class 2 BRAF-L597R Mutant

Data for paper published in Biomolecules June 2022  Isothermal Titration Calorimetry results for CDC37-BRAF interactions. dil in the filename donates the heat of dilution. Heats of dilution are in to buffer. Pairs with the same date donate a set of experiments. After the date the interacting partner proteins or small molecule is shown. Use Origin program to access the data files. Abstract: The kinome specific co-chaperone, CDC37, is responsible for delivering BRAF to the Hsp90 complex, where it is then translocated to the RAS complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognising the C-lobe of BRAF kinase domain, while the C-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF.  We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signalling in a clinical setting, particularly for class 2 BRAF mutations. </p

Thermal Shift Assay for BRAF mutants for paper: Recognition of BRAF by CDC37 and Re-evaluation of the Activation mechanism for the Class 2 BRAF-L597R Mutant

Data for paper published in Biomolecules June 2022  Raw data for Thermal shift asay of BRAF mutants. Use the LightCycler 480 SW 1.5 software or similar to access the data files. Abstract: The kinome specific co-chaperone, CDC37, is responsible for delivering BRAF to the Hsp90 complex, where it is then translocated to the RAS complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognising the C-lobe of BRAF kinase domain, while the C-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF.  We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signalling in a clinical setting, particularly for class 2 BRAF mutations. </p

PyMol cartoon showing dimerization of AIP TPR-domain through crystal lattice contacts.

<p>(A), The AIP domains are in green and yellow. Amino acid residues are in magenta or cyan, hydrogen bonds as blue dotted lines, water molecules as red spheres and bound TOMM20 AQSLAEDDVE-peptide used in the crystallization in gold. However, only residues AEDDVE are visible in the structure. The TPR domains are symmetrically related and hydrogen bonding is shown in only one half of the figure. The cartoon shows that Arg 304 is hydrogen bounded directly to the neighboring TOMM20 bound peptide (gold). (B), PyMol cartoon showing a close up of the main interactions between Arg 304 and bound peptide used in the crystallization (AQLSLAED₃D₄VE) in panel A. However, only residues AED₃D₄VE are visible in the structure. (C), Co-immunoprecipitation of Flag-AIP and Myc-AIP in the presence of TOMM20 peptide (AQSLAEDDVE). The results show that Flag-AIP and Myc-AIP do not co-immunoprecipitate. M, molecular mass markers, with molecular mass indicated to the left of the panel; lane 1 and 5 AIP input (cleared lysate) protein; lane 2 and 6 are anti-Myc co-immunoprecipitation, lanes 3 and 7 are anti-Flag co-immunoprecipitations, while lanes 4 and 8 are IgG control. Lanes 1–4 (first gel) was blotted for Myc tag and lanes 5–8 (second gel) for Flag tag. The arrow indicates the position where the flag- and myc-tagged AIP runs (40 Kd). (D), The core interaction of the AIP dimerization interface shows that E192 is buried and shielded from solvent by Ala 312, Arg 188 and Trp 279.</p

PyMOL diagram showing binding interactions.

<p>(A) Interactions with HSP90β EDASRMEEVD peptide and (B), with TOMM20 AQSLAEDDVE peptide bound to the TPR domain of AIP. Peptide residues that where visible (SRMEEVD and AEDDVE) are shown in red as single letter code. Dotted blue lines represent hydrogen bonds and green, the amino acid residues involved; red-colored spheres, water molecules and yellow residues, residues solely in van der Waals contact. The structures were obtained at 2.0 (PDB, 4AIF) and 1.9 Å (PDB, 4APO), respectively. (C), Molecular switching in the TPR domain of AIP. The alternative conformations of Lys 266 allow selection of the Hsp90 MEEVD- (green) or TOMM20 EDDVE-motif (cyan). Dotted blue lines represent hydrogen bonds while red-colored spheres represent water molecules.</p

PyMol diagram showing the conservation of residues on the surface of AIP TPR-domain.

<p>The most highly conserved residues line the cavity of the TPR domain in which the TPR-motif containing peptides bind to.</p

Sequence conservation of the Cα-7h of AIP.

<p>(A), sequence alignment showing conservation of amino acid residues. Ss, Salmo salar (NM_001140060.1); Dr, Danio rerio (NM_214712.1); Rn, Rattus norvegicus (NM_172327.2); Mm, Macaca mulatta (NM_001194313); Ca, Chlorocebus aethiops (O97628); Hs, Homo sapiens (FJ514478.1); Bt, Bos taurus (NM_183082.1), Xt, Xenopus (Silurana) tropicalis (NM_001102749.1) and Cc, Caligus clemensi (BT080130.1). (I313⁺), Ile 313 represents the last residue in the sequence that is involved in packing interactions of the TPR domain. Mutations associated with disease are indicated above the sequence. (* below the sequence), Amino acids at these positions are identical; (:), highly conserved (.) or conserved. Arg 304 of Human AIP is shown in red type face. Numbers above the sequence (positions 1 to 15) represent residue numbers of the helical wheel shown in panel B. (B), Helical wheel showing the position of identical and conserved residues form the alignment in panel A for the Cα-7h of AIP. Orange, non-polar; green, polar uncharged; pink, acidic and blue, basic amino-acid residues. (C), PyMol cartoon showing a hypothetical helix (residues beyond Arg 325) with the identical and highly conserved amino acid residues shown in panels A and B. Conserved residues on one side of the helix are shown in green and on the other in yellow. Residue numbers shown are those in panel B, while those in brackets are actual residue numbers in panel A. (D), The TPR-domain of the R304* mutant of AIP. Deletion of the terminal region of AIP (transparent helical region) allows chaperone binding but disrupts association with PDE4A5 and AhR.</p

PyMol cartoon of the structure of human AIP.

<p>(A), PyMol cartoon of the HSP90β EDASRMEEVD-peptide (green) bound to the TPR domain of AIP (cyan). Only SRMEEVD of the peptide was visible. The structure was obtained at 2.0 Å (PDB, 4AIF) while that with the TOMM20 AQSLAEDDVE-peptide was obtained at 1.9 Å (PDB, 4APO, not shown). The A and B helices of each TPR motif (TPR1 to 3) and the C-terminal alpha helix (α-7) are indicated. (B), Superimposition of peptide conformations of HSP90β EDASRMEEVD (green), TOMM20 AQSLAEDDVE (cyan) bound to AIP (only SRMEEVD and AEDDVE of the peptides is shown), and HSP90α DTSRMEEVD (yellow) peptide bound to CHIP, showing that the peptide backbone conformation is essentially the same.</p

Binding of peptide to the TPR domains of Hop and AIP.

<p>(A), PyMol Space-filling model showing the binding of the MEEVD peptide of HSP90 to the TPR domain of Hop TPR2A and (B), the EDASRMEEVD peptide of HSP90β bound to the TPR domain of AIP (only SRMEEVD of the peptide is shown). (C), Superimposition of the peptides bound to the TPR domains of HOP2A (yellow) and AIP (green).</p