The ISWI family protein SMARCA5 contains the ATP-binding pocket that coordinates the catalytic Mg2+ ion and water molecules for ATP hydrolysis. In this study, we demonstrate that SMARCA5 can also possess an alternative metal-binding ability. First, we isolated SMARCA5 on the cobalt column (IMAC) to near homogeneity. Examination of the interactions of SMARCA5 with metal-chelating supports showed that, apart from Co2+, it binds to Cu2+, Zn2+ and Ni2+. The efficiency of the binding to the last-listed metal was influenced by the chelating ligand, resulting in a strong preference for Ni-NTA over the Ni-CM-Asp equivalent. To gain insight in the preferential affinity for the Ni-NTA ligand, QM calculations were performed on model systems and metal-ligand complexes with a limited protein fragment of SMARCA5 containing the double-histidine (dHis) motif. The calculations correlated the observed affinity with the relative stability of the d-block metals to tetradentate ligand coordination over tridentate, as well as their overall octahedral coordination capacity. Likewise, binding free energies derived from model imidazole complexes mirrored the observed Ni-NTA/Ni-CM-Asp preferential affinity. Finally, similar calculations on complexes with a SMARCA5 peptide fragment derived from the AlphaFold structural prediction, captured almost accurately the expected relative stability of the TM complexes, and produced a large energetic separation (~10 kcal∙mol-1) between Ni-NTA and Ni-CM-Asp in favour of the former.

• MeSH
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové metabolismus   chemie   MeSH
• kovy chemie   metabolismus   MeSH
• kvantová teorie MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• restrukturace chromatinu MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.

• MeSH
• chromatografie afinitní metody   MeSH
• krevní proteiny analýza   chemie   metabolismus   MeSH
• lidé MeSH
• magnety MeSH
• mikrosféry MeSH
• molekulární modely MeSH
• myosin typu I chemie   metabolismus   MeSH
• prognóza MeSH
• roztroušená skleróza krev   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• vazba proteinů MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH