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

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

Scientific reports 2021 Sep 22

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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. © 2021. The Author(s).

Citation

Zachary M Huttinger, Laura M Haynes, Andrew Yee, Colin A Kretz, Matthew L Holding, David R Siemieniak, Daniel A Lawrence, David Ginsburg. Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape. Scientific reports. 2021 Sep 22;11(1):18827