Topology of the stable serpin-protease complexes revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin.
Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P'1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.
Picard
V
V
INSERM, Unité 428, Université Paris V, 75270 Paris Cedex 06, France.
Marque
P E
PE
Paolucci
F
F
Aiach
M
M
Le Bonniec
B F
BF
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001323:Autoantibodies / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*