Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy

Abstract

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R, Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Analyzing Protein Disorder with IUPred2A

“…The corresponding region also has experimentally solved structures, as shown in the PDB line with a green box. The N-terminal region of the protein is predicted as disordered, indicated by the high IUPred values (red line), in agreement with experimental characterization (Delaforge et al, 2018). Disordered binding regions predicted by ANCHOR2 are shown by the blue line.…”

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex

“…To further define the dynamic interface of Gcn4, we measured 15 N-relaxation parameters for nAD and nAD:ABD1. An increase in the transverse relaxation rate, R 2 (= 1/T 2 ), upon binding is indicative of a role in binding for the affected residues ( Delaforge et al, 2018 ). Upon binding ABD1, each of the hydrophobic regions of nAD shows an increase in R 2 , supporting the direct involvement of these regions in binding ABD1 ( Figures 2E and S3C ).…”

15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

“…68,69 and non-local and local dynamics of IDPs using mainly15 N CPMG based relaxation dispersion experiments[15][16][17][18]67,[69][70][71][72][73][74][75][76][77][78][79][80] . Several experimental strategies have been designed to allow the recording of 201 H-15 N correlation spectra81,82 and CPMG relaxation experiments of IDPs under physiological conditions and obviate the influence of amide exchange43,83 , but the adverse impact of D 2 O through the isotope effect has to our knowledge escaped attention.…”