Fig. 6: Probing the structural role of metal ions and cofactors in GLT25D1/COLGALT1 GT1 and GT2 domains.
a DSF analysis of wild-type GLT25D1 (left) incubated with Mn2+ (purple trace) shows significant stabilization induced by binding of the metal ion when compared to untreated samples (blue trace); removal of amino acid side chains critical for binding of metal ions in the GT2 domain such as Glu435 (center) and Asp437 (right) results in loss of the metal ion-induced stabilization effect. b DSF analysis of GLT25D1/COLGALT1 subject to EDTA (green trace) and EGTA (orange trace) treatment, showing the destabilization induced by metal ion chelation when compared to untreated samples (blue trace); Comparison between wild type (left) and mutants in the GT2 domain highlight very similar destabilization effects. c EDTA-treated samples show alterations in dispersity when tested using mass photometry. d EDTA-treated samples show altered elution profiles when tested using size-exclusion chromatography. e Native-MS spectra under partially-denaturing conditions of wild-type 30+ charge state (left) and Trp158Arg 31+ charge state (center), with the signals corresponding to the free protein and protein-ligand complex labeled; box plot (bounds: percentile 25–75%; mean value: black line; whiskers: maxima-minima) of ligand mass calculation obtained from the entire charge state distribution (n = 6 for wild-type, n = 7 for Trp158Arg) of Native-MS spectra (right); reference lines report the theoretical ligand mass as a function of distinct divalent cation cofactors.
