Deciphering the Multisite Interactions of a Protein and Its Ligand at Atomic Resolution by Using Sensitive Paramagnetic Effects

• Published in: Chemistry : a European journal 2017-01, Vol.23 (4), p.926-934
• Main Authors: Ma, Fei‐He, Wang, Xiao, Chen, Jia‐Liang, Wen, Xin, Sun, Han, Su, Xun‐Cheng
• Format: Article
• Language: English
• Subjects: Affinity; Animals; Binding; Binding Sites; Chemistry; Chickens; Coordination Complexes - chemistry; Coordination Complexes - metabolism; Flexibility; Lanthanoid Series Elements - chemistry; Ligands; Magnetics; Molecular Dynamics Simulation; molecular recognition; multisite interactions; Muramidase - chemistry; Muramidase - metabolism; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; paramagnetic effects; Protein Binding; Protein Structure, Tertiary; Proteins; protein–ligand complexes; Simulation; Spectrum analysis; Water - chemistry
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201604393

Quantitative analysis of multisite interactions between a protein and its binding partner at atomic resolution is complicated because locating the binding sites is difficult and differentiating the flexibility of each binding site is even more elusive. Introduction of a paramagnetic metal center close to the binding pocket greatly attenuates the signals in the NMR spectrum upon binding. Herein, the multisite binding of hen egg white lysozyme (HEWL) with lanthanide complexes [Ln(DPA)3]3− (DPA=dipicolinic acid) was analyzed with sensitive paramagnetic NMR spectroscopy. Paramagnetic relaxation enhancement (PRE) revealed that HEWL interacts with [Ln(DPA)3]3− at four major binding sites in aqueous solution, which is in contrast to a previous X‐ray structural analysis. The varied binding affinities for the ligands and different flexibilities at each binding site were in good agreement with atomistic molecular dynamics (MD) simulations. The present work demonstrates that a combination of paramagnetic NMR spectroscopy and MD simulations is a powerful tool to delineate the multisite interactions of a protein with its binding partner at atomic resolution, in terms of both affinity and flexibility. Good combination: The combination of paramagnetic NMR spectroscopy and MD simulations is a powerful technique to delineate multisite interactions between a protein and a ligand. This method allows quantitative analysis of the binding affinities and flexibility of the protein–ligand complex (see figure), which can be helpful for drug design.