Low Resolution X-ray Structure of Human Methylamine-treated α2-Macroglobulin

Abstract

The structure of methylamine-treated human ␣ 2 -macroglobulin (␣ 2 M-MA), a 720-kDa tetrameric inactivated proteinase inhibitor from plasma, has been determined to a resolution of 10 Å. Data were collected with synchrotron radiation at 120 K, and phases were calculated by multiple isomorphous replacement and solvent flattening. A novel feature of the structure of ␣ 2 M is present in its proteinase-binding cavity, dividing it into two compartments. The potential sites for proteinase entrapment in these compartments are sterically restricted. The positions of the thiol groups appearing from the functional important thiol esters upon their cleavage have been determined. They are found at the walls of the compartments at the center of the structure. The overall structure of ␣ 2 M-MA is much more sphere-like than previously inferred from electron microscopy studies. However, several aspects of the structure are well described by recent three-dimensional reconstructions. Possible models for the monomer, the disulfide bridged dimer, and native ␣ 2 M are discussed.1 is the best studied member of the class of proteinase-binding ␣-macroglobulins (for reviews, see Refs. 1-3). One subunit of ␣ 2 M contains 1451 residues of which eight are glycosylated. This subunit has a mass of 180 kDa (4). Two subunits form disulfide bridged dimers (5). Two such dimers make noncovalent contacts to form the 720-kDa functional tetramer.The native form of ␣ 2 M can form complexes with various proteinases. This complex formation is initiated by specific limited proteolysis of the bait region (6) found at residues 667-705 (7). The cleavage of the bait region initiates a series of conformational changes in the ␣ 2 M subunits resulting in entrapment of the attacking proteinase inside the tetramer. The final result of these changes is the transformed form of ␣ 2 M. The native and transformed forms of ␣ 2 M appear in electrophoresis as the slow and fast forms of ␣ 2 M (6, 8). The native form of ␣ 2 M contains internal ␤-Cys-␥-Glu thiol esters, formed from Cys-949 and Glu-952 in each subunit. During complex formation, these thiolesters become activated, and this activation results in covalent binding of the proteinase primarily through ⑀-Lys(proteinase)-␥-Glu(␣ 2 M) cross-links (9 -11). The bound proteinase is still active, but it is only accessible to small substrates and inhibitors. Two small proteinase molecules the size of chymotrypsin, but only one large proteinase like plasmin, can be bound to ␣ 2 M (12). A final result of the conformational change is that sites in the C-terminal domains (residues 1314 -1451) (13-15) become exposed for interaction with the cellular receptor for ␣ 2 M-proteinase complexes. This receptor has been found to be identical to the low density lipoprotein receptor-related protein (16 -18). Incubation of ␣ 2 M with methylamine also leads to thiol ester cleavage and covalent binding of methylamine (9,19). The conformation of the resulting molecule, ␣ 2 M-MA, resembles that of the fast form ␣ 2 M-proteinase complex (20, ...

The Binding of Receptor-recognized α2-Macroglobulin to the Low Density Lipoprotein Receptor-related Protein and the α2M Signaling Receptor Is Decoupled by Oxidation

“…The carboxyl-terminal 20 kDa of ␣ 2 M is partially exposed in the native conformation (45), and therefore, it is not unexpected that it would be partially recognized by a polyclonal antisera to RBF. Upon exposure of native ␣ 2 M to proteinase or methylamine, this region becomes more exposed so that it binds to cell surface receptors and should, therefore, show greater recognition by antibodies raised against RBF.…”

Identical or Overlapping Sequences in the Primary Structure of Human α2-Macroglobulin Are Responsible for the Binding of Nerve Growth Factor-β, Platelet-derived Growth Factor-BB, and Transforming Growth Factor-β

“…The target peptide bonds are located within a small region in the center of the ␣ 2 M subunit called the "bait domain" (4,6). Proteinase-induced bait domain cleavage causes conformational change in ␣ 2 M and a change in the size and shape of the central cavity (7)(8)(9). As a result, reacting proteinases are irreversibly trapped within the ␣ 2 M cylinder (1,4).…”

Mechanical unfolding of a2‐macroglobulin molecules with atomic force microscope

“…Its amino acid sequence is known [13] and the low resolution crystallographic analysis of a chemically treated form has recently reported [14]. The native protein has a diameter of 20-30 nm under an electron microscope [12].…”