Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries.
Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition of the unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degrees C, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose, a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degrees C, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life of PAI-1 245-fold. Furthermore, within the limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to vitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g. the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separated.
Stoop
A A
AA
Department of Biochemistry Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Eldering
E
E
Dafforn
T R
TR
Read
R J
RJ
Pannekoek
H
H
D000818:Animals; D016384:Consensus Sequence; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019096:Vitronectin; D010959:Tissue Plasminogen Activator