The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted.
Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.
Hägglöf
Peter
P
Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Bergström
Fredrik
F
Wilczynska
Malgorzata
M
Johansson
Lennart B-A
LB
Ny
Tor
T
D000818:Animals; D001665:Binding Sites; D001896:Boron Compounds; D004220:Disulfides; D004735:Energy Transfer; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004220:Disulfides; D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D012996:Solutions