Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.
The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.
Wardell
M R
MR
Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. mrw@patmos.wustl.edu
Chang
W S
WS
Bruce
D
D
Skinner
R
R
Lesk
A M
AM
Carrell
R W
RW
D000595:Amino Acid Sequence; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism
D058833:Antithrombin Proteins; D000991:Antithrombins; C108837:L-antithrombin; D009842:Oligopeptides; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D012697:Serine Endopeptidases; D013917:Thrombin; D019791:Guanidine