Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape

Zachary M Huttinger,Daniel A Lawrence,Colin A Kretz,Laura M Haynes,David R Siemieniak,David Ginsburg,Matthew L Holding,Andrew Yee

doi:10.1038/s41598-021-97871-7

Abstract

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure–function relationships for PAI-1 and for the interpretation of human genomic sequence variants.

Highlights

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator
Selection of phage displayed PAI-1 in the presence of a ninefold molar excess of a negative control, phage displaying the A3 domain of von Willebrand Factor (VWF, a protein fragment not known to interact with uPA, phVWF-A3), for complex formation with uPA (Fig. 2) resulted in at least a five-fold enrichment in phPAI-1 relative to phVWF-A3, indicating that the immunoprecipitation is specific for uPA and uPA:PAI-1 complexes
The phPAI-1 mutant library exhibits a depth of 8.04 × 1 06 independent clones with an average of 4.2 ± 1.8 amino acid substitutions/molecule as determined by Sanger sequencing of 13 randomly selected phage clones

Summary

Introduction

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). If the rate of RCL insertion is slower than the resolution of the acyl intermediate, complete proteolysis of the scissile bond occurs, with the SERPIN functioning as a substrate instead of an inhibitor[9] Either of these mechanisms is possible when a SERPIN interacts with a given protease. The overall goal is to map the mutational landscape of PAI-1 with respect to mutations that render PAI-1 no longer functional to (1) better understand the natural evolution of PAI-1 with respect to the amino acid space that can be occupied at any given residue, and (2) to use this high resolution map of PAI-1 as a basis for engineering novel SERPINs. SERPINs, including PAI-1, have been previously engineered to inhibit proteases other than their canonical targets[26,27] demonstrating the potential to develop novel therapeutics for the treatment of a variety of disorders including hemophilia and alpha-1-antitrypsin deficiency. PAI-1 is a attractive choice as a SERPIN scaffold as it lacks native cysteine residues and remains functional in the absence of glycosylation, facilitating large-scale production of functionally active, recombinant PAI-1 in bacterial s ystems[28,29]

Methods

Results

Conclusion