Localization of basic residues required for receptor binding to the single α-helix of the receptor binding domain of human α2-macroglobulin

To better understand the structural basis for the binding of proteinase-transformed human alpha 2-macrogrobulin (alpha(2)M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha(2)M. Assignment of the backbone NMR resonances of RED was made using C-13/N-15 and N-15-enriched RED expressed in Escherichia coli. The secondary structure of RED was determined using H-1 and C-13 chemical shift indices and inter- and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein. The beta-strands farm three regions of antiparallel beta-sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378). Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.