Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma

Tapon‐Bretaudière, Jacqueline; Bros, A.; Couture‐Tosi, Evelyne; Delain, Etienne

doi:10.1002/j.1460-2075.1985.tb02321.x

Cited by 61 publications

(43 citation statements)

References 23 publications

(14 reference statements)

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“…A significant contribution to the understanding of tihe structural forms seen by electron microscopy has been reported by Tapon-Bretaudiere et al (16). These authors suggested that the so-called open and closed forms of a2M were in fact the same structure viewed from the side and the end, respectively.…”

mentioning

confidence: 95%

“…This discrepancy seems to have resulted from the study of a2M that had undergone proteolysis during its isolation (16). Early studies reported the presence of two forms (7,(17)(18)(19): an ovate structure shaped like two crescents facing each other with a bar in the center (the "closed" form) and a larger structure, conventionally described as a cyrillic X (the "opened" form).…”

mentioning

confidence: 99%

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Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Bretaudiere

Tapon‐Bretaudière

Stoops

1988

Proc. Natl. Acad. Sci. U.S.A.

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Well-preserved structures of native and achymotrypsin-bound a2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native a2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the a2-macroglobulin-chymotrypsin conijugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of a2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex. a2-Macroglobulin (a2M) is one of the major antiproteases found in the plasma of vertebrates. It has the capacity to inhibit not only endoproteases normally present in the plasma but also proteases from other origins (1). a2M, isolated from human plasma, is a large glycoprotein (Mr, 725,000) composed of four identical subunits (Mr, 180,000) (2, 3). The quaternary structure consists of two noncovalently bound protomers; each protomer is made up of two subunits covalently linked by two disulfide bonds (4). When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5-7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11). The inactivated protease may form a covalently bound complex with a2M by reacting with an intramolecular thiol ester linkage between glutamine and cysteine residues. Even though there are four "bait" sites available, it has been proposed that a2M has two independent and identical proteinase binding sites (12)(13)(14). By using energy transfer experiments, Pochon et al. (12) demonstrated that the two sites are 4.4 nm apart. The binding of 2 mol of proteases per mol of a2M has been observed only with smaller proteases like trypsin and chymotrypsin (Mr, 25,000); larger proteases like plasmin (Mr, 81,000) display a 1:1 molar binding ratio (15).There have been conflicting reports relating the various structures of a2M obtained by electron microscopy to the native and protease bound forms. This discrepancy seems to have resulted from the study of a2M that had undergone proteolysis during its isolation (16). Early studies reported the presence of two forms (7,(17)(18)(19): an ovate structure shaped like two crescents facing each other with a bar in the center (the "closed" form) and a ...

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mentioning

confidence: 95%

mentioning

confidence: 99%

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Bretaudiere

Tapon‐Bretaudière

Stoops

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Its dimensions are 180 -200 Å, 120 -140 Å, and 80 -90 Å as estimated from projections of different orientations and from three-dimensional reconstructions (25)(26)(27)(28)(29)(30)(31)(32)(33). Receptor recognition sites are located at the tip of each of the arms of the H (27,34).…”

mentioning

confidence: 99%

“…Receptor recognition sites are located at the tip of each of the arms of the H (27,34). When studied by EM, native ␣ 2 M has various shapes, which among others resemble a twisted cross (3,26), a doughnut (25), and a padlock (26,35). It has recently been shown that all of these shapes correspond to one single structure (36).…”

mentioning

confidence: 99%

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

Andersen

Koch

Dolmer

et al. 1995

Journal of Biological Chemistry

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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, ...

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“…In both processes Cys-SH groups appear. In electron micrographs native ~M resembles irregular donuts [3][4][5][6]. After reaction with proteinases or methylamine, c~2M has a more compact H-like shape with the proteinase(s) occupying a central elongated cavity [5][6][7][8][9][10].…”

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confidence: 99%

Crystallization and preliminary X‐ray analysis of methylamine‐treated α₂‐macroglobulin and 3 α₂‐macroglobulin‐proteinase complexes

et al. 1991

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Crystals of methylamine.treated a2-macroglobulin (¢E2M-MA), cz2-macroglobulin in complex with two molecules of trypsin, ~2M-T2, one molecule of plasmin, e~2M-PL, and one molecule of plasmin followed by methylamine.treatrnent, ~zM-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of a2M-MA diffracted to at least 9 A resolution, crystals off,M-T2 diffracted to 10 A resolution and crystals of~zM-PL and a~M-PL(MA) diffracted to 11 ~. resolution. For ~M-MA th~ cell parameters were determined as: a=b=257 A, c=555 A; and for ~M-T2 as: a=b=247 A, c=559 A. For both preparations the spaea group was I4(1)22. As estimated from density measurements, the crystals of ~zM-MA and r~2M-T2 contain one 360 kDa ~M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, V,, was estimated at 5.6 A~/Da. The crystal parameters of ~M-PL and ~M-PL(MA) were not determined.

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Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma

Cited by 61 publications

References 23 publications

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

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

Crystallization and preliminary X‐ray analysis of methylamine‐treated α₂‐macroglobulin and 3 α₂‐macroglobulin‐proteinase complexes

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Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma

Cited by 61 publications

References 23 publications

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

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

Crystallization and preliminary X&#x2010;ray analysis of methylamine&#x2010;treated α2&#x2010;macroglobulin and 3 α2&#x2010;macroglobulin&#x2010;proteinase complexes

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Crystallization and preliminary X‐ray analysis of methylamine‐treated α₂‐macroglobulin and 3 α₂‐macroglobulin‐proteinase complexes