Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'.


Abstract

The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively. The micromolar affinity calcium binding site (site A) has been deleted (Delta75-83) in these variants, enabling the activity and thermostability measurements in chelating conditions. Each of the variants includes mutations known previously to increase the thermostability of calcium-independent subtilisin in addition to new stabilizing mutations. S63 has eight amino acid replacements: D41A, M50F, A73L, Q206W, Y217K, N218S, S221C, and Q271E. S63 has 75-fold greater stability than wild type subtilisin in chelating conditions (10 mm EDTA). The other variant, S88, has ten site-specific changes: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, and Q271E. The two new cysteines form a disulfide bond, and S88 has 1000 times greater stability than wild type subtilisin in chelating conditions. Comparisons of the two new crystal structures (S63 in space group P2(1) with A cell constants 41.2, 78.1, 36.7, and beta = 114.6 degrees and S88 in space group P2(1)2(1)2(1) with cell constants 54.2, 60.4, and 82.7) with previous structures of subtilisin BPN' reveal that the principal changes are in the N-terminal region. The structural bases of the stabilization effects of the new mutations Q2K, S3C, P5S, D41A, Q206C, and Q206W are generally apparent. The effects are attributed to the new disulfide cross-link and to improved hydrophobic packing, new hydrogen bonds, and other rearrangements in the N-terminal region. Study holds ProTherm entries: 15453 Extra Details: calcium binding site; activity; thermostability; hydrophobic packing

Submission Details

ID: vYyZDFdB3

Submitter: Connie Wang

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

Version: 1

Publication Details
Almog O;Gallagher DT;Ladner JE;Strausberg S;Alexander P;Bryan P;Gilliland GL,J. Biol. Chem. (2002) Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'. PMID:12011071
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
1A2Q 1998-01-08T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 7186
1AK9 1997-05-30T00:00:00+0000 1.8 SUBTILISIN MUTANT 8321
1AQN 1997-07-31T00:00:00+0000 1.8 SUBTILISIN MUTANT 8324
1AU9 1997-09-12T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 8324 IN CITRATE
1DUI 2000-01-17T00:00:00+0000 2.0 Subtilisin BPN' from Bacillus amyloliquefaciens, crystal growth mutant
1GNS 2001-10-06T00:00:00+0000 1.8 SUBTILISIN BPN'
1GNV 2001-10-10T00:00:00+0000 1.9 CALCIUM INDEPENDENT SUBTILISIN BPN' MUTANT
1LW6 2002-05-30T00:00:00+0000 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1S01 1989-08-21T00:00:00+0000 1.7 LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING
1S02 1991-02-20T00:00:00+0000 1.9 EFFECTS OF ENGINEERED SALT BRIDGES ON THE STABILITY OF SUBTILISIN BPN'

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Subtilisin BPN' P00782 SUBT_BACAM