Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.


Abstract

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.

Submission Details

ID: airXUDgq3

Submitter: Shu-Ching Ou

Submission Date: Dec. 5, 2018, 5:16 p.m.

Version: 1

Publication Details
Lawrence DA;Olson ST;Palaniappan S;Ginsburg D,J Biol Chem (1994) Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. PMID:7961684
Additional Information

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
395.6 A,B,C,D Plasminogen activator inhibitor 1 P05121 PAI1_HUMAN