Low-resolution X-ray diffraction data obtained from hexagonal crystals of methylamine-treated α2-macroglobulin

Andersen, Gregers Rom; Thirup, Søren; Nyborg, Jens; Dolmer, Klavs; Jacobsen, Linda; Sottrup‐Jensen, Lars

doi:10.1107/s0907444993013125

Cited by 3 publications

(5 citation statements)

References 10 publications

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“…The map contoured at 1 showed a large part of the solvent region occupied by low density. From this map, it was evident that the asymmetric unit contained one monomer of ␣ 2 M-MA or three molecules in the unit cell and not one dimer as previously stated (45).…”

Section: Methodssupporting

confidence: 54%

“…Assuming that the thiol groups are far more reactive than other nucleophilic groups, the occupancy for substitution at the thiol groups is 78.5%. Crystals of the TAMM derivative of ␣ 2 M-MA were grown as described previously (45). Because the crystals of ␣ 2 M-MA are not stable for prolonged time in protein-free solution, the two other derivatives were prepared by adding solid Ta 6 Br 14 (49) or PIP (48) directly to the drops such that solid material was still present during the whole soaking period of 3-4 days.…”

Section: Methodsmentioning

confidence: 99%

“…The data were indexed, integrated, and scaled as described previously (45), except that the program INST_HAM-BURGD (50) was used for autoindexing. Completeness was 95% for native data, 79% for the TAMM derivative, 80% for the Ta 6 Br 14 derivative, and 70% for the PIP derivative.…”

Section: Methodsmentioning

confidence: 99%

“…Tetragonal bipyramidal crystals of ␣ 2 M-MA and of several ␣ 2 M-proteinase complexes diffracting to a maximum of 9 -11 Å resolution were reported earlier (44). Hexagonal crystals of ␣ 2 M-MA were recently found to be suitable for structural investigations (45), although the limited diffraction power would not allow a detailed structural analysis. Crystals of ␣ 2 M-MA from other species (46) and of the homologous complement component C3 (47) show similar resolution limits even with the use of high intensity synchrotron radiation and data collection at cryogenic temperature.…”

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confidence: 99%

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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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Section: Methodssupporting

confidence: 54%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…on 7.5% slab gels according to the procedure of Laemmli [10]. Although an X-ray structure of the conformationally altered form of ~2M has recently been determined [3], the resolution…”

Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Bait region‐thiol ester mapping in human α₂‐macroglobulin

et al. 1995

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Materials and methodsAbstract The separations between aromatic residues in the bait region and nitroxide spin labels attached to the thiol ester-form-2.1. Isolation of human plasma ~2M ing residues (Cys 949 and Gin 9s2) in human a2-macroglobulin Plasma ~2M was isolated from outdated pooled human plasma, ob-(a2M) have been determined from paramagnetic broadening el'-tained from Vanderbilt University Hospital Blood Bank, by affinity fects of the spin labels on bait region lH NMR signals. We found chromatography on zinc chelate resin using the procedure of Imber and that both the Cys 949 and Gin 9s2 residues are within 11-17 A of the Pizzo [5]. The ~zM was judged to be homogeneous by SDS-PAGE. ~2 M concentrations were determined spectrophotometrically using an ex° aromatic residues in the bait region, with closer approach of some tinction coefficient of 640,000 M i . cm i at 280 nm [6]. (1H) groups was measured by DTNB assay [7] immediately prior to reaction with the labelling reagent and gave close to the theoretical value of 4 SH groups per ~2M tetramer. Covalent attachment of the amino-TEMPO label to the Gin 952 side chain was by direct reaction of native ~2M with 500 mM amino-TEMPO, resulting in attack of the amino group on the 1. Introduction thiolestercarbonyl. Because ofthe bulk ofamino-TEMPO the reaction was very slow compared to reaction with the much smaller methylamine Cleavage of the bait region in human ~2 M by proteinase is [8]. The progress of the reaction was therefore checked periodically by native gel electrophoresis, looking for conversion from slow to fast the initiating event that results in the large-scale conformational form. After 7 days the reaction was judged, by this criterion, to be >95% changes that trap the proteinase and lead to binding of the complete. All samples were dialyzed extensively to remove unreacted ~2M-proteinase complex to receptor through exposure of the free nitroxide. receptor binding region on the ~2M [1]. The mediator of the conformational changes is an internal thiol ester which stabi- Polyacrylamide gel electrophoresisNon-denaturing PAGE was carried out on 5% acrylamide slab gels lizes the native conformation of the ~2M. Bait region cleavage according to the procedure of Davis [9]. SDS-PAGE was carried out results in activation and rapid opening of the thiol ester [2]. on 7.5% slab gels according to the procedure of Laemmli [10]. Although an X-ray structure of the conformationally altered form of ~2M has recently been determined [3], the resolution EPR spectroscopyEPR spectra were recorded at 277K on a Bruker ESP300 specis too low to permit tracing of the polypeptide chain and thus trometer operating at 9.8 GHz, using a field set of 3490 G, a scan range localization of particular regions of the protein. We therefore of 100 G and a modulation frequency of 100 kHz. Each spectrum was still do not have a good idea of the structural relationship of the average of 10 scans. Sample concentrations were between 14 and the bait region relative to the thiol ester. The only d...

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