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
Aa. Stoop et al., 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, J MOL BIOL, 305(4), 2001, pp. 773-783
Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine pr
otease inhibitor (serpin) superfamily. Its highly mobile reactive-center lo
op (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) conform
ation (t(1/2) at 37 degreesC, 2.2 hours). We determined the amino acid resi
dues responsible for the inherent instability of PAI-1, to assess whether t
hese properties are independent and, consequently, whether the structural b
asis for inhibition and latency transition is different. For that purpose,
a hypermutated PAI-1 library that is displayed on phage was pre-incubated f
or increasing periods (20 to 72 hours) at 37 degreesC, prior to a stringent
selection for rapid t-PA binding. Accordingly, four rounds of phage-displa
y 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-contribu
ting 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 muta
nts isolated are functionally indistinguishable from wild-type PAI-1 with r
espect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to
vitronectin. These stabilizing mutations constitute largely reversions to t
he stable "serpin consensus sequence" and are located in areas implicated i
n PAI-1 stability (e.g, the vitronectin-binding domain and the proximal hin
ge). Collectively, our data provide evidence that the structural requiremen
ts for PAI-1 loop insertion during latency transition and target proteinase
inhibition can be separated. (C) 2001 Academic Press.