Mapping protein-protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy.

Abstract

REDOR-based experiments with simultaneous 1H-13C and 1H-15N dipolar dephasing are explored for investigating intermolecular protein-protein interfaces in complexes formed by a U-13C,15N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U-13C,15N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by 1H-13C or 1H-15N cross polarization, permitting to establish the residues of the U-13C,15N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into 13C-13C correlation experiments. We established the validity of this approach on U-13C,15N-histidine and on a structurally characterized complex of dynactin's U-13C,15N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.