STD and WLOGSY NMR Based Fingerprinting Reveals Subtle and Biologically Relevant Differences in Short Linear Motif Binding

Abstract

Interactions between Short Linear Motifs (SLiMs) and a partner domain are commonly exploited as simplified functional models of transient Protein-Protein Interactions (PPI) for characterizing interfacial associations between partner proteins. In this study, we report the use of a ligand-observed NMR approach, where through unambiguous assignment of 1H resonances of two closely related SLiMs, whilst bound to their partner domain (HopTPR2A ), we could assign STD and WLOGSY NMR signals to specific regions in the peptide backbone. These data revealed subtle alterations in magnetization transfer, resulting from changes in the binding mode of each SLiM respectively. The ability to detect and compare these changes at sub-residue resolution, provided differing fingerprints of SLiM binding. This approach therefore represents a broadly accessible method for identifying binding hot spots and interrogating the impact of structural variations on SLiM-domain interaction stability, and by extension transient PPIs.