Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability.


Abstract

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin. Study holds ProTherm entries: 16601, 16602, 16603 Extra Details: 0.1mM EDTA and 0.1% (w/v) PEG 6000 were added in the experiment. shutter region; conformational changes; D-helix; converse effect

Submission Details

ID: WAkPHCh3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Quinsey NS;Fitton HL;Coughlin P;Whisstock JC;Dafforn TR;Carrell RW;Bottomley SP;Pike RN,Biochemistry (2003) Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability. PMID:12939144
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
3KCG 2010-02-02 1.7 Crystal structure of the antithrombin-factor IXa-pentasaccharide complex
2B5T 2006-09-19 2.1 2.1 Angstrom structure of a nonproductive complex between antithrombin, synthetic heparin mimetic SR123781 and two S195A thrombin molecules
1TB6 2004-08-17 2.5 2.5A Crystal Structure of the Antithrombin-Thrombin-Heparin Ternary Complex
1NQ9 2003-09-30 2.6 Crystal Structure of Antithrombin in the Pentasaccharide-Bound Intermediate State
1E04 2000-06-02 2.6 PLASMA BETA ANTITHROMBIN-III
2ANT 1997-06-16 2.6 THE 2.6 A STRUCTURE OF ANTITHROMBIN INDICATES A CONFORMATIONAL CHANGE AT THE HEPARIN BINDING SITE
1JVQ 2003-06-03 2.6 Crystal structure at 2.6A of the ternary complex between antithrombin, a P14-P8 reactive loop peptide, and an exogenous tetrapeptide
1E05 2000-06-02 2.62 PLASMA ALPHA ANTITHROMBIN-III
1OYH 2004-04-13 2.62 Crystal Structure of P13 Alanine Variant of Antithrombin
1R1L 2004-10-05 2.7 Structure of dimeric antithrombin complexed with a P14-P9 reactive loop peptide and an exogenous tripeptide (formyl-norleucine-LF)
2BEH 2005-11-01 2.7 Crystal structure of antithrombin variant S137A/V317C/T401C with plasma latent antithrombin
1T1F 2005-10-04 2.75 Crystal Structure of Native Antithrombin in its Monomeric Form
1LK6 2003-06-03 2.8 Structure of dimeric antithrombin complexed with a P14-P9 reactive loop peptide and an exogenous tripeptide
2ZNH 2008-10-21 2.8 Crystal Structure of a Domain-Swapped Serpin Dimer
2B4X 2006-10-10 2.8 Crystal Structure of Antithrombin-III
1DZG 2000-05-26 2.8 N135Q-S380C-ANTITHROMBIN-III
4EB1 2012-07-18 2.8 Hyperstable in-frame insertion variant of antithrombin
1DZH 2000-05-26 2.85 P14-FLUORESCEIN-N135Q-S380C-ANTITHROMBIN-III
1AZX 1999-01-13 2.9 ANTITHROMBIN/PENTASACCHARIDE COMPLEX
1BR8 1998-09-02 2.9 IMPLICATIONS FOR FUNCTION AND THERAPY OF A 2.9A STRUCTURE OF BINARY-COMPLEXED ANTITHROMBIN
1E03 2000-06-02 2.9 PLASMA ALPHA ANTITHROMBIN-III AND PENTASACCHARIDE
1ANT 1995-01-26 3.0 BIOLOGICAL IMPLICATIONS OF A 3 ANGSTROMS STRUCTURE OF DIMERIC ANTITHROMBIN
3EVJ 2008-10-21 3.0 Intermediate structure of antithrombin bound to the natural pentasaccharide
1SR5 2004-08-17 3.1 ANTITHROMBIN-ANHYDROTHROMBIN-HEPARIN TERNARY COMPLEX STRUCTURE
1ATH 1995-02-07 3.2 THE INTACT AND CLEAVED HUMAN ANTITHROMBIN III COMPLEX AS A MODEL FOR SERPIN-PROTEINASE INTERACTIONS
2GD4 2006-05-09 3.3 Crystal Structure of the Antithrombin-S195A Factor Xa-Pentasaccharide Complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 ANTITHROMBIN Q5R5A3 ANT3_PONAB
100.0 Antithrombin-III P01008 ANT3_HUMAN