In vitro and in silico design of alpha1-antitrypsin mutants with different conformational stabilities.


Abstract

Alpha(1)-antitrypsin, a protein belonging to the serine protease inhibitor (serpin) superfamily, is characterized by the ability to undergo dramatic conformational changes leading to inactive polymers. Serpin polymerization, which causes a range of diseases such as emphysema, thrombosis and dementia, occurs through a process in which the reactive center loop residues of one serpin molecule insert into the A beta-sheet of another. PoPMuSiC, a program that uses database-derived mean force potentials to predict changes in folding free energy resulting from single-site mutations, was used to modulate rationally the polymerization propensity of alpha(1)-antitrypsin. This was accomplished by generating mutants with a stabilized active form and destabilized polymerized form, or the converse. Of these mutants, five were expressed and characterized experimentally. In agreement with the predictions, three of them, K331F, K331I and K331V, were shown to stabilize the active form and decrease the polymerization rate, and one of them, S330R, to destabilize the active form and to increase polymerization. Only one mutant (K331T) did not display the expected behavior. Thus, strikingly, the adjacent positions 330 and 331, which are located at the beginning of the beta-strand next to the additionally inserted beta-strand in the polymerized form, have opposite effects on the conformational change. These residues therefore appear to play a key role in inducing or preventing such conformational change. Study holds ProTherm entries: 15788, 15789, 15790, 15791, 15792, 15793, 15794, 15795, 15796, 15797, 15798, 15799, 15800, 15801, 15802, 15803, 15804, 15805 Extra Details: Native -> Intermediate in silico design; serpin; conformational disease; sequence design; protein stability

Submission Details

ID: w8vBmdiC4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Gilis D;McLennan HR;Dehouck Y;Cabrita LD;Rooman M;Bottomley SP,J. Mol. Biol. (2003) In vitro and in silico design of alpha1-antitrypsin mutants with different conformational stabilities. PMID:12498804
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
1ATU 1997-05-11T00:00:00+0000 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
1D5S 1999-10-11T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
1D5S 1999-10-11T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
1EZX 2000-05-12T00:00:00+0000 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1EZX 2000-05-12T00:00:00+0000 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1HP7 2000-12-12T00:00:00+0000 2.1 A 2.1 ANGSTROM STRUCTURE OF AN UNCLEAVED ALPHA-1-ANTITRYPSIN SHOWS VARIABILITY OF THE REACTIVE CENTER AND OTHER LOOPS
1IZ2 2002-09-19T00:00:00+0000 2.2 Interactions causing the kinetic trap in serpin protein folding
1KCT 1996-08-06T00:00:00+0000 3.46 ALPHA1-ANTITRYPSIN
1OO8 2003-03-03T00:00:00+0000 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
1OPH 2003-03-05T00:00:00+0000 2.3 NON-COVALENT COMPLEX BETWEEN ALPHA-1-PI-PITTSBURGH AND S195A TRYPSIN

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