Advanced techniques of solution nuclear magnetic resonance spectroscopy for structural investigation of protein-protein interaction

Abstract

Investigation of the protein-protein interaction mode at atomic resolution is essential for understanding on the underlying functional mechanisms of proteins as well as for discovering druggable compounds blocking deleteriou interprotein interactions. Solution NMR spectroscopy provides accurate and precise information on intermolecular interactions even for weak and transient interactions, and it is also markedly useful for examining the change in the conformation and dynamics of target proteins upon binding events. In this mini-review, we comprehensively describe three unique and powerful methods of solution NMR spectroscopy, paramagnetic relaxation enhancement (PRE), pseudo-contact shift (PCS), and residual dipolar coupling (RDC), for the study on protein-protein interactions.

Figure 1. Illustration of paramagnetic effect-based NMR methods for investigation of protein-protein interactions. Effects of paramagnetic relaxation enhancement (PRE) (A) and pseudo-contact shift (PCS) (B) are shown. The alteration of the intensity (A) and chemical shift (B) of NMR signals of isotopically-labeled protein are analysed. PRE causes attenuation of the signal intensity depending on the distance, and PCS induces chemical shift changes depending on the distance and angle from the immobilized paramagnetic center (small red sphere). The region in which PRE and PCS, which affects the NMR signal, is highlighted with gray spheres with dotted line and four ellipses, respectively. Black spheres with numbering (in the target proteins) indicate the location of the residues, which are corresponding to the NMR spectra.

Figure 2. Schematic illustration of residual dipolar coupling (RDC) experiments. (A) Orientation of the dipole-dipole vector in the target protein in the cartesian space is determined. (B) Schematic representation of the two-dimensional ¹H-¹⁵N spectrum for RDC. The “Aligned” and “Isotropic” (reference experiment) show peak splitting in the presence and absence of orienting media, respectively. J_HN and D indicate the constants of J-coupling and RDC, respectively. The asterisk indicates the chemical shift alteration in the midpoint of the split signals (denoted as small circles), which results from the PCS effect when the alignment is achieved by lanthanide ions.