Amino acid selective cross-saturation method for identification of proximal residue pairs in a protein-protein complex

We describe an NMR-based approach, the amino acid selective cross-saturation (ASCS) method, to identify the pairs of the interface residues of protein-protein complexes. ASCS uses a "cross-saturation (CS)-donor" protein, in which only one amino acid is selectively ¹H-labeled in a ²H-background, and a "CS- acceptor" protein with uniform ²H,¹⁵N labeling. Irradiation of the ¹H-labeled amino acid, which exists only in the donor, decreases the intensity of the ¹H-¹⁵N HSQC signals of the acceptor residues proximal to the ¹H-labeled CS-source residue(s) through the CS phenomenon. Given the three-dimensional structure of each protein in the complex, but not the complex structure, the combinatorial analysis of multiple ASCS results specify the CS-source residue(s), based on the spatial complementarity between the CS-source residues on the CS donor and the cross-saturated amide protons on the acceptor. NMR investigations of the labeling selectivity and efficiency in an E. coli host, which are critical for ASCS, revealed that Ala, Arg, His, Ne, Leu, Lys, Met, Phe, Pro, Trp, and Tyrare selectively labeled with a high ¹H/²H ratio. The observation of the ASCS was then confirmed using the known structure of the yeast ubiquitin (Ub) and yeast ubiquitin hydrolase 1 (YUH1). Conversely, reasonable candidates for the CS-source residues were suggested by the analysis of the ASCS results, with reference to the individual structures of YUH1 and Ub. The pairwise distance information between the CS-source residues and the cross-saturated amide groups obtained by ASCS will be useful for modeling protein-protein complexes.