NMR Methods for Structural Characterization of Protein-Protein Complexes

Abstract

Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here we discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.

An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening

“…Pools containing one (or more) ligand(s) of the receptor protein generate shifts of the NMR signals that are easily observable by overlaying the measured HSQC spectra ( Fig. 2 c) ( Purslow et al, 2020 ). To facilitate acquisition and analysis of the NMR data, CSP experiments were measured on 15 N-labeled samples of the isolated N- (EIN) and C-terminal (EIC) domains of EI that, being considerably smaller than the full-length protein, generate highly resolved NMR spectra characterized by high signal-to-noise ratio.…”

Current Methods for Detecting Cell Membrane Transient Interactions

“…In comparison, nuclear magnetic resonance (NMR) spectroscopy is better for analyzing structure and dynamics of membrane transient complexes (Díaz-Moreno et al, 2005 ; Dannatt et al, 2016 ; Purslow et al, 2020 ). Thermodynamic information such as the binding affinities of membrane partners can also be measured in NMR (Purslow et al, 2020 ). NMR is a powerful technique for studying membrane transient interactions, especially if atomic-level structural information is needed.…”

“…However, these methods are normally not suitable for studying transient interactions or conformational changes. In comparison, nuclear magnetic resonance (NMR) spectroscopy is better for analyzing structure and dynamics of membrane transient complexes (Díaz-Moreno et al, 2005;Dannatt et al, 2016;Purslow et al, 2020). Thermodynamic information such as FIGURE 2 | The use of NMR and mass spectrometry for studying membrane transient interactions.…”

“…the binding affinities of membrane partners can also be measured in NMR (Purslow et al, 2020). NMR is a powerful technique for studying membrane transient interactions, especially if atomiclevel structural information is needed.…”

Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR

“…Chemical shift perturbation and changes in linewidth are some of the commonly used techniques in studies of protein–ligand interaction and can serve as tools for ligand screening and drug discovery [ 10 ]. Both solution NMR and solid-state NMR can be used to characterise protein–protein interactions and complex structures, using a variety of techniques such as chemical shift perturbation, solvent paramagnetic relaxation enhancement and heteronuclear dipolar recoupling [ 11 ].…”