Analysis of thiolester bond cleavage-dependent conformational changes in binary α 2-macroglobulin-proteinase complexes

Abstract

The structures of the two proteinase-binding sites in human α 2-macroglobulin (α 2M) were probed by treatment of α 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with α 2M (a binary α 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in α 2M. Binary α 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary α 2M-proteinase complexes with CH 3NH 2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in α 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the α 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol α 2M and completion of the conformational change in the complex. Similar treatment of the α 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol α 2M. Unlike the α 2M-thrombin complex, the α 2M-plasmin complex did not undergo a complete conformational change following treatment with CH 3NH 2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1–4 region of the α 2M-bound plasmin heavy chain, and following this treatment the α 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary α 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the α 2M-plasmin complex, however, the heavy chain of α 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.