map3k6 tap  (Addgene inc)

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: ASK1, ASK2 and ASK3 C-terminal domains have different oligomerisation propensity. (A) Overview of domain architecture, and conservation of ASK1–3. (B) Size-exclusion chromatography of ASK1(1290–1374), ASK2(1216–1288) and ASK3(1241–1313), corresponding to regions of respective ASK proteins labelled ‘SAM’ domain in panel A. (C) Sedimentation velocity AUC analysis of ASK1, 2 and 3 domains at indicated concentrations spanning between 0.15 and 3.0 mg/mL (15–365 µM).

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Size-exclusion Chromatography, Sedimentation

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Structure of the ASK3 SAM domain. (A) Cartoon representation of the crystal structure of ASK3(1290-1374) displaying the three monomers within the asymmetric unit. The ML-EH interface is indicated with a dashed box. (B) Alignment of SAM domains of human ASK1–3. (C) Close-up view of wild-type residues within the dashed areas of the ML-EH interface. (D) Size-exclusion chromatography trace indicating the disruption of the wild-type ASK3-SAM oligomer as a result of a aspartate to lysine mutation at position 1279 (ASK3(D1279K)). (E) SEC-MALS data measuring the molar mass of a cysteine to glutamate mutant at position 1291 (ASK3(C1291E)), relative to the wild-type oligomer.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Size-exclusion Chromatography, Disruption, Mutagenesis

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: ASK3 SAM crystal packing. Cartoon Illustration of the crystallographic asymmetric unit, and one SAM domain from a neighbouring asymmetric unit that interacts at the α1/α2 interface. Interfaces referred to in the text are indicated

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques:

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Analysis of ASK3 SAM mutants (A) closeup view of residues at the C-terminal interface. (B) Size-exclusion chromatography of the WT ASK3 SAM and mutations at the C-terminal (left) and α1/α2 (right) interface.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Potential ASK1 oligomer interfaces. (A) Size-exclusion chromatography of WT ASK1 SAM and F1369Q mutation (equivalent to ASK3 Y1300Q). (B) Closeup view of the ASK3 C-terminal tail interaction, relative to sequence conservation in ASK1. Indicated on the alignment are ASK1/ASK3 residues F1369/Y1300, Lys1372/Ala1303 (which would could not be accomodated in a putative ASK1 complex), and Thr1374/Glu1305, to indicate the the position of the final residue of ASK1. (C) SEC-MALLS of WT and C1360E mutation of ASK1. (D) SEC MALS of WT ASK1, ASK2 and a mixture of the two SAM domains, each at a total concentration of 200 µM.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Size-exclusion Chromatography, Mutagenesis, Sequencing, Residue, Concentration Assay

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Heterotypic interactions between ASK SAM domains (A) GST-pulldown experiments measuring the ability of GST-ASK1, -ASK2, and -ASK3 SAM domains to pull down untagged SAM domains from ASK1, ASK2 and ASK3 respectively (B) Sedimentation velocity analytical ultracentrifugation of the isolated SAM domains of ASK1, ASK2 (1.5 mg/mL) and an equimolar mixture of the two (at 1.5 mg/mL each). (C) GST-pulldown experiments measuring the ability of WT GST-ASK1 and GST-ASK2 SAM domains to pull down either WT, or Cysteine mutant ASK1 and ASK2 SAM domains. (D) Analytical size-exclusion chromatography comparing the ability of WT and C1360E ASK1 SAM domain to form a higher-order oligomer with the WT ASK2 SAM domain. (E) Analytical size-exclusion chromatography comparing the ability of WT and C1268E ASK2 SAM domain to form a higher-order oligomer with the WT ASK1 SAM domain.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Sedimentation, Analytical Ultracentrifugation, Isolation, Mutagenesis, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Comparisons of the ML-EH pairwise interaction of ASK3 with indicated SAM ML-EH structures.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques:

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: The ASK SAM domain ML-EH interface (A) Left: Schematic view of the ML-EH interface. Right: Electrostatic surfaces of ASK1–3 SAM domains (as calculated using APBS (37), with regions predicted to participate in ML-EH contacts outlined in yellow. Models of ASK1 and ASK2 were generated using MODELLER, based on the ASK3 SAM domain solved here. (B) Illustration of the ML-EH interface seen within the ASK3 asymmetric unit (ML-EH) and the similar but slightly offset arrangement with a crystallographically related SAM domain (ML-EH*) (C) Comparison of the ML-EH and ML-EH* interfaces. Pairs of SAM domains participating in each type of interface overlaid based on the bottom SAM domain. The top SAM domain is offset, quantitated by an 18° shift of the α5 helix.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Generated, Comparison

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: SAXS analysis of ASK1+2 and ASK3 SAM domains. (A)Schematic illustrating the helicies formed from the different ML-EH interfaces seen in the crystal lattice. (B/C) Experimental scattering curves with the best CRYSOL modelled fit (black line) and the Gunier plot (inset) for, ASK1+2 SAM (B); and ASK3 SAM (C). (C/D) Distance distribution plots for, ASK1+2 SAM (D); and ASK3 SAM (E). (F/G) Side and top views of best fit models for the (F) ASK1+2 SAM hexamer (alternating ML-EH/ML-EH*) and (G) ASK3 SAM hexamer (repeated ML-EH; within asymmetric unit). (H)Summary table of the fit of each model to the experimental SAXS data.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques:

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Comparison of SAXS models to scattering data. CRYSOL fits of models to experimental SAXS scattering data, with corresponding model illustrated below for (A/B) ASK1 SAM; (C) ASK2 SAM; (D–G) ASK1+2 SAM; (H–L) ASK3 SAM.

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Comparison

Journal: bioRxiv

Article Title: Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

doi: 10.1101/693663

Figure Lengend Snippet: Comparison of the pitch of helical filaments formed by SAM domain ML-EH interfaces. (A) Putative ASK1-ASK2 alternating ML-EH:ML-EH*. (B) Putative ASK3 repeating ML-EH. (C) SHANK3 from Rattus norvegicus (PDB ID: 2F3N). (D) Human ETS-related protein TEL1 (PDB ID: 1JI7). (E) Human Diacylgylcerol kinase 1 (PDB ID: 3BQ7). (F) Human Tankyrase 2 (PDB ID: 5JRT).

Article Snippet: Constructs comprising ASK2 and ASK3 were amplified from Addgene plasmids (#69727 and #69728, respectively).

Techniques: Comparison