Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method

Abstract

Rationale Interactions of drug molecules and proteins play important roles in physiological and pathological processes in vivo. It is of significance to establish a reliable strategy for studying protein–drug ligand interactions and would be helpful for the design and screening of new drugs in pharmacological research. Methods The interactions between four indole alkaloids (IAs) extracted from Ophiorrhiza japonica (O. japonica) and myoglobin (Mb) protein were investigated using a multi‐spectrometric and computational method of native electrospray ionization mass spectrometry (native ESI‐MS), hydrogen/deuterium exchange mass spectrometry (HDX‐MS), circular dichroism (CD) and molecular docking (MD). Results The IA‐bound Mb complexes were analyzed using native ESI‐MS, with the obtained protein‐to‐ligand stoichiometry at 1:1, 1:2 and 1:3. Binding constants were measured according to the interpretation of MS spectra. MD complemented MS measurements, probing the binding sites and modes of the four IAs to Mb. Analyses involving CD and HDX‐MS demonstrated that exposure to IAs could affect the conformation of Mb by decreasing the α‐helix content and made Mb more susceptible to HDX at the backbone. Conclusions A new MS‐based integrated analysis method has been developed to successfully study the interactions of Mb and IAs extracted from O. japonica. The experimental and calculation results have good consistency, revealing all of the four IA molecules could bind to Mb to form 1:1, 1:2 and 1:3 Mb–IA complexes. The order of binding ability of these IAs to Mb was ophiorrhine B > compound C > ophiorrhine A > compound D. CD and HDX‐MS results indicated that binding with IAs destabilizes Mb. HDX‐MS analysis suggests that Mb becomes more susceptible to HDX, indicating that binding with IAs destabilizes the structure of Mb. In addition, the interaction with IAs affected the overall structure of Mb, ascribed to the decrease of α‐helix content and less folding of the backbone.

Recent advances in proteomics and metabolomics in plants

“…Notably, the recent development of a MS-cleavable cross linkers such as disuccinimidyl sulfoxide (Kao et al 2011) and disuccinimidyl dibutyric urea (DSBU) (Ihling et al 2020) allows to cleave cross-linked peptides during MS/MS for subsequent MS3 acquisition of cleaved peptides, which facilitates peptide identification using traditional database based approaches (Liu et al, 2015) and allows multiplexed quantitative XL-MS (Yu et al 2016). In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is able to determine the interaction surfaces and solvent-exposed regions and is emerging as a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein-small molecule interactions (Masson et al 2019;Li et al 2020d;Gutkowska et al 2021).…”