Localization of basic residues required for receptor binding to the singlealpha-helix of the receptor binding domain of human alpha(2)-macroglobulin

To better understand the structural basis for the binding of proteinase-tra nsformed human alpha 2-macrogrobulin (alpha(2)M) to its receptor, we have u sed three-dimensional multinuclear NMR spectroscopy to determine the second ary structure of the receptor binding domain (RBD) of human alpha(2)M. Assi gnment 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 consis ts of eight strands in beta-conformation and one alpha-helix, which togethe r comprise 44% of the protein. The beta-strands farm three regions of antip arallel 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 str ucture predictions of other alpha-macroglobulins show the conservation of t his alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.