Localization of basic residues required for receptor binding to the single α‐helix of the receptor binding domain of human α ₂ ‐macroglobulin

To better understand the structural basis for the binding of proteinase‐transformed human α₂‐macroglobulin (α₂M) to its receptor, we have used three‐dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human α₂M. Assignment of the backbone NMR resonances of RBD was made using ¹³C/¹⁵N and ¹⁵N‐enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using ¹H and ¹³C chemical shift indices and inter‐ and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in β‐conformation and one α‐helix, which together comprise 44% of the protein. The β‐strands form three regions of antiparallel β‐sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the α‐helix, which also contains an (Arg1378). Secondary structure predictions of other α‐macroglobulins show the conservation of this α‐helix and suggest an important role for this helix and for basic residues within it for receptor binding.