Probing the role of the F-helix in serpin stability through a single tryptophan substitution.


Abstract

Serpins form loop-sheet polymers through the formation of a partially folded intermediate. Through mutagenesis and biophysical analysis, we have probed the conformational stability of the F-helix, demonstrating that it is almost completely unfolded in the intermediate state. The replacement of Tyr160 on the F-helix of alpha1-antitrypsin to alanine results in the loss of a conserved hydrogen bond that dramatically reduces the stability of the protein to both heat and solvent denaturation, indicating the importance of Tyr160 in the stability of the molecule. The mutation of Tyr160 to a tryptophan residue, within a fluorescently silent variant of alpha1-antitrypsin, results in a fully active, stable serpin. Fluorescence analysis of the equilibrium unfolding behavior of this variant indicates that the F-helix is highly disrupted in the intermediate conformation. Iodide quenching experiments demonstrate that the tryptophan residue is exposed to a similar extent in both the intermediate and unfolded states. Cumulatively, these data indicate that the F-helix plays an important role in controlling the early conformational changes involved in alpha1-antitrypsin unfolding. The implications of these data on both alpha1-antitrypsin function and misfolding are discussed. Study holds ProTherm entries: 14925, 14926, 14927, 14928, 14929, 14930, 14931, 14932, 14933, 14934, 14935, 14936, 14937, 14938, 14939, 14940, 14941, 14942, 14943 Extra Details: partially folded intermediate; conformational stability; hydrogen bond; tryptophan

Submission Details

ID: 3jfJvPFC4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Cabrita LD;Whisstock JC;Bottomley SP,Biochemistry (2002) Probing the role of the F-helix in serpin stability through a single tryptophan substitution. PMID:11926819
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
3NDD 2010-07-28 1.5 Cleaved antitrypsin with P10 Pro, and P9-P6 Asp
5NBU 2018-03-21 1.67 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
5NBV 2018-03-21 1.73 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
3NE4 2011-12-14 1.81 1.8 Angstrom structure of intact native wild-type alpha-1-antitrypsin
4PYW 2015-06-10 1.91 1.92 angstrom crystal structure of A1AT:TTAI ternary complex
1QLP 1999-09-27 2.0 2.0 ANGSTROM STRUCTURE OF INTACT ALPHA-1-ANTITRYPSIN: A CANONICAL TEMPLATE FOR ACTIVE SERPINS
2QUG 2008-08-12 2.0 Crystal structure of alpha-1-antitrypsin, crystal form A
1HP7 2001-03-14 2.1 A 2.1 ANGSTROM STRUCTURE OF AN UNCLEAVED ALPHA-1-ANTITRYPSIN SHOWS VARIABILITY OF THE REACTIVE CENTER AND OTHER LOOPS
1IZ2 2003-02-11 2.2 Interactions causing the kinetic trap in serpin protein folding
3DRM 2009-03-31 2.2 2.2 Angstrom Crystal Structure of Thr114Phe Alpha1-Antitrypsin
1OPH 2003-08-05 2.3 NON-COVALENT COMPLEX BETWEEN ALPHA-1-PI-PITTSBURGH AND S195A TRYPSIN
3CWL 2008-09-23 2.44 Crystal structure of alpha-1-antitrypsin, crystal form B
3CWM 2008-09-23 2.51 Crystal structure of alpha-1-antitrypsin complexed with citrate
1EZX 2000-10-25 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1QMB 2000-02-06 2.6 Cleaved alpha-1-antitrypsin polymer
1OO8 2003-08-05 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
1ATU 1997-08-20 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
3NDF 2010-07-28 2.7 Cleaved antitrypsin with P8-P6 Asp
1PSI 1996-12-07 2.92 Intact recombined alpha1-antitrypsin mutant PHE 51 to LEU
1D5S 2000-04-02 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
7API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
9API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
8API 1990-10-15 3.1 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
3DRU 2009-03-31 3.2 Crystal Structure of Gly117Phe Alpha1-Antitrypsin
2D26 2005-11-29 3.3 Active site distortion is sufficient for proteinase inhibit second crystal structure of covalent serpin-proteinase complex
5IO1 2016-06-08 3.34 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN Z ALPHA-1-ANTITRYPSIN
1KCT 1997-01-11 3.46 ALPHA1-ANTITRYPSIN
3T1P 2011-08-17 3.9 Crystal structure of an alpha-1-antitrypsin trimer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.1 Alpha-1-antitrypsin O00394 A1AT_CHLAE
92.4 Alpha-1-antitrypsin P01010 A1AT_PAPAN
96.4 Alpha-1-antitrypsin Q5RCW5 A1AT_PONAB
100.0 Alpha-1-antitrypsin P01009 A1AT_HUMAN