Deciphering the Multisite Interactions of a Protein and Its Ligand at Atomic Resolution by Using Sensitive Paramagnetic Effects.
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.
State Key Laboratory of Elemento-Organic Chemistry and Collaborative, Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
Fei-He
FH
Ma
State Key Laboratory of Elemento-Organic Chemistry and Collaborative, Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
Xiao
X
Wang
State Key Laboratory of Elemento-Organic Chemistry and Collaborative, Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
Jia-Liang
JL
Chen
State Key Laboratory of Elemento-Organic Chemistry and Collaborative, Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
Xin
X
Wen
Department of Structural Biology, Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Str. 10, 13125, Berlin, Germany.
Han
H
Sun
State Key Laboratory of Elemento-Organic Chemistry and Collaborative, Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
Xun-Cheng
XC
Su
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D056831:Coordination Complexes / Q000737:chemistry / Q000378:metabolism; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008024:Ligands; D008280:Magnetics; D056004:Molecular Dynamics Simulation; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014867:Water / Q000737:chemistry
D056831:Coordination Complexes; D028581:Lanthanoid Series Elements; D008024:Ligands; D014867:Water; C421864:hen egg lysozyme; D009113:Muramidase
NMR spectroscopy; molecular recognition; multisite interactions; paramagnetic effects; protein-ligand complexes