Journal: Advanced Science
Article Title: Mortalin and PINK1/Parkin‐Mediated Mitophagy Represent Ovarian Cancer‐Selective Targets for Drug Development
doi: 10.1002/advs.202505592
Figure Lengend Snippet: Identification of mortalin amino acids that interact with mortalin and confirmation of SHetA2 binding to mortalin substrate binding domain (SBD) (A). Mortalin protein structure with a DDK tag used in the NMR analysis. B) Chemical structure of SHetA2 and points of interaction with mortalin amino acids identified by NMR. The colored arrows and text refer to the amino acids identified to bind SHetA2 as shown in panels (G–K). C) Percentage reflectivity changes (∆% R ), of the indicated concentrations of SHetA2 binding to surface immobilized full‐length mortalin. D) Line profile for the binding interactions in panel C (inset shows the 3D representations of the surface plasmon resonance (SPR) imager spots). E) ∆% R of SHetA2 with surface immobilized mortalin SBD. F) Line profile for the binding interactions shown in panel E (inset shows the 3D representations of the SPR imager spots). G) NMR chemical shift perturbation (difference between free and bound states), [22] shows amino acid sites affected by the interaction with SHetA2; the program VMD is used for visualization. [23] Binding of SHetA2 (H–K), and an analog (L), with the substrate binding domain of mortalin. H ) NMR signals with over 0.2 ppm perturbation highlighted on the crystal structure and indicating specific mortalin SBD amino acids that interact with SHetA2. I) Two SHetA2 molecules docked onto the crystal structure of mortalin (PDB 3N8E), with the lowest energy −8.5 kcal mol −1 . J) Another view of panel D showing additional hydrophobic interactions. K) Another docked configuration with −7.7 kcal mol −1 binding energy. L) Structure of SHetA2 analog with longer linker and its binding to mortalin.
Article Snippet: Antibodies, anti‐Pink1 (#6946), ‐PARKIN (#4211), ‐Cox‐IV (#4850), ‐BNIP3 (#3769), ‐TOM20 (#42406S), ‐p62 (#5114s), ‐αTibulin (#2125S), ‐BCL‐XL (2764S#), ‐BCL2 (4223S#), ‐LC3 I/II (#12741S), ‐Beclin‐1 (#3738s), ‐DDK (#14793s), ‐IP3R (#8568) and ‐Mortalin Grp75 (D13H4)‐XP (#3593s), anti‐GAPDH (#5174), anti‐β‐actin (#4970), secondary antibodies conjugated with horse radish peroxidase (HRP), anti‐mouse (#7076S) or anti‐Rabbit (#7074V), Scrambled or Beclin1 siRNA (#6222) were purchased from cell signaling technology (Danvers, MA, USA).
Techniques: Binding Assay, SPR Assay
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![Identification <t>of</t> <t>mortalin</t> amino acids that interact with mortalin and confirmation of SHetA2 binding to mortalin substrate binding domain (SBD) (A). Mortalin protein structure with a <t>DDK</t> tag used in the NMR analysis. B) Chemical structure of SHetA2 and points of interaction with mortalin amino acids identified by NMR. The colored arrows and text refer to the amino acids identified to bind SHetA2 as shown in panels (G–K). C) Percentage reflectivity changes (∆% R ), of the indicated concentrations of SHetA2 binding to surface immobilized full‐length mortalin. D) Line profile for the binding interactions in panel C (inset shows the 3D representations of the surface plasmon resonance (SPR) imager spots). E) ∆% R of SHetA2 with surface immobilized mortalin SBD. F) Line profile for the binding interactions shown in panel E (inset shows the 3D representations of the SPR imager spots). G) NMR chemical shift perturbation (difference between free and bound states), [22] shows amino acid sites affected by the interaction with SHetA2; the program VMD is used for visualization. [23] Binding of SHetA2 (H–K), and an analog (L), with the substrate binding domain of mortalin. H ) NMR signals with over 0.2 ppm perturbation highlighted on the crystal structure and indicating specific mortalin SBD amino acids that interact with SHetA2. I) Two SHetA2 molecules docked onto the crystal structure of mortalin (PDB 3N8E), with the lowest energy −8.5 kcal mol −1 . J) Another view of panel D showing additional hydrophobic interactions. K) Another docked configuration with −7.7 kcal mol −1 binding energy. L) Structure of SHetA2 analog with longer linker and its binding to mortalin.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9392/pmc12499392/pmc12499392__ADVS-12-e05592-g006.jpg)