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

Abstract

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.