Molecular evolution of plasminogen activator inhibitor-1 functional stability.


Abstract

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) supergene family and a central regulatory protein in the blood coagulation system. PAI-1 is unique among serpins in exhibiting distinct active and inactive (latent) conformations in vivo. Though the structure of latent PAI-1 was recently solved, the structure of the short-lived, active form of PAI-1 is not known. In order to probe the structural basis for this unique conformational change, a randomly mutated recombinant PAI-1 expression library was constructed in bacteriophage and screened for increased functional stability. Fourteen unique clones were selected, and shown to exhibit functional half-lives (T1/2S) exceeding that of wild-type PAI-1 by up to 72-fold. The most stable variant (T1/2 = 145 h) contained four mutations. Detailed analysis of these four mutations, individually and in combination, demonstrated that the markedly enhanced functional stability of the parent compound mutant required contributions from all four substitutions, with no individual T1/2 exceeding 6.6 h. The functional stability of at least eight of the remaining 13 compound mutants also required interactions between two or more amino acid substitutions, with no single variant increasing the T1/2 by > 10-fold. The nature of the identified mutations implies that the unique instability of the PAI-1 active conformation evolved through global changes in protein packing and suggest a selective advantage for transient inhibitor function.

Submission Details

ID: jwTQqwyw

Submitter: Shu-Ching Ou

Submission Date: Nov. 21, 2018, 11:55 a.m.

Version: 1

Publication Details
Berkenpas MB;Lawrence DA;Ginsburg D,EMBO J (1995) Molecular evolution of plasminogen activator inhibitor-1 functional stability. PMID:7621813
Additional Information

Study Summary

Number of data points 62
Proteins Plasminogen activator inhibitor 1
Unique complexes 31
Assays/Quantities/Protocols Experimental Assay: Half-Life (37°C)
Libraries Mutants of PAI-1

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1A7C 1999-03-23 1.95 HUMAN PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 IN COMPLEX WITH A PENTAPEPTIDE
3CVM 2008-08-19 2.02 High resolution structure of a stable Plasminogen activator inhibitor type-1 in its protease cleaved form
1DVN 2000-09-13 2.1 LATENT FORM OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1)
4G8R 2013-12-25 2.19 Crystal Structure of a novel small molecule inactivator bound to plasminogen activator inhibitor-1
1OC0 2003-06-19 2.28 plasminogen activator inhibitor-1 complex with somatomedin B domain of vitronectin
3PB1 2010-12-29 2.3 Crystal Structure of a Michaelis Complex between Plasminogen Activator Inhibitor-1 and Urokinase-type Plasminogen Activator
3Q02 2011-06-22 2.3 Crystal structure of plasminogen activator inhibitor-1 in a metastable active conformation.
4IC0 2013-12-11 2.32 Crystal Structure of PAI-1 in Complex with Gallate
4AQH 2012-11-28 2.4 Plasminogen activator inhibitor type-1 in complex with the inhibitor AZ3976
1DVM 2000-09-06 2.4 ACTIVE FORM OF HUMAN PAI-1
3UT3 2013-02-06 2.42 A novel PAI-I inhibitor and its structural mechanism
1C5G 1999-12-17 2.6 PLASMINOGEN ACTIVATOR INHIBITOR-1
3EOX 2009-02-17 2.61 High quality structure of cleaved PAI-1-stab
3Q03 2011-06-22 2.64 Crystal structure of plasminogen activator inhibitor-1 in a metastable active conformation.
3R4L 2011-11-02 2.7 Human very long half life Plasminogen Activator Inhibitor type-1
1DB2 1999-11-10 2.7 CRYSTAL STRUCTURE OF NATIVE PLASMINOGEN ACTIVATOR INHIBITOR-1
9PAI 1999-03-19 2.7 CLEAVED SUBSTRATE VARIANT OF PLASMINOGEN ACTIVATOR INHIBITOR-1
4G8O 2013-12-25 2.71 Crystal Structure of a novel small molecule inactivator bound to plasminogen activator inhibitor-1
6I8S 2019-02-13 2.9 Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1
1B3K 1999-12-11 2.99 Plasminogen activator inhibitor-1
5BRR 2015-09-02 3.16 Michaelis complex of tPA-S195A:PAI-1
5ZLZ 2019-04-03 3.58 Structure of tPA and PAI-1

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Plasminogen activator inhibitor 1 P05121 PAI1_HUMAN