The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation.
A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.
Qazi
U
U
Dept. of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, TX 77030, USA.
Kolodziej
S J
SJ
Gettins
P G
PG
Stoops
J K
JK
D019943:Amino Acid Substitution / Q000235:genetics*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D006801:Humans; D008744:Methylamines / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*
D004952:Esters; D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine; D010450:Endopeptidases; D003545:Cysteine