Thermodynamic analysis of the structural stability of the shiga toxin B-subunit.


Abstract

The conformational stability of Shiga toxin B-subunit (STxB), a pentameric protein from Shigella dysenteriae has been characterized by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy under different solvent conditions. It is shown that the thermal folding/unfolding of STxB is a reversible process involving a highly cooperative transition between folded pentamer and unfolded monomers. The conformational stability of STxB is pH dependent and because of its pentameric nature is also concentration dependent. STxB is maximally stable in the pH range from 5 to 9 (Delta G upon unfolding is close to 13 kcal per mol of monomer at 25 degrees C), and its stability decreases both at lower and at higher pH values. The pH dependence of the Gibbs energy of stabilization between pH 2.5 and 5 is consistent with the change in the ionizable state of an average of four groups per monomer upon unfolding. Structural thermodynamic calculations show that the stabilization of the STxB pentamer is primarily due to the interactions established between monomers rather than intramonomer interactions. The folding of an isolated monomer into the conformation existing in the pentamer is unfavorable and expected to be characterized by a free-energy change upon folding in the order of 2.5 kcal mol(-1) at 25 degrees C. On the average, intersubunit interaction induced upon oligomerization of folded monomers should contribute close to -13.4 kcal per mol of monomer to bring the overall Gibbs energy to the experimentally determined value at this temperature. Study holds ProTherm entries: 16553, 16554, 16555, 16556, 16557, 16558, 16559, 16560, 16561, 16562, 16563, 16564, 16565, 16566, 16567, 16568, 16569, 16570, 16571, 16572, 16573, 16574 Extra Details: pentameric protein; cooperative transition; concentration dependent; intramonomer interactions

Submission Details

ID: gw24GH7M3

Submitter: Connie Wang

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

Version: 1

Publication Details
Pina DG;Gómez J;Villar E;Johannes L;Shnyrov VL,Biochemistry (2003) Thermodynamic analysis of the structural stability of the shiga toxin B-subunit. PMID:12899637
Additional Information

Study Summary

Number of data points 44
Proteins Shiga toxin subunit A ; Shiga toxin subunit B
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp buffers:Phosphate: 10 mM, prot_conc:34.7 microM ; Experimental Assay: dHvH prot_conc:34.7 microM ; Experimental Assay: dG buffers:Phosphate: 10 mM, prot_conc:34.7 microM ; Experimental Assay: dCp buffers:Phosphate: 10 mM, prot_conc:25.5 microM ; Experimental Assay: dHvH prot_conc:25.5 microM ; Experimental Assay: dG buffers:Phosphate: 10 mM, prot_conc:25.5 microM ; Experimental Assay: dCp buffers:Phosphate: 10 mM, prot_conc:18.6 microM ; Experimental Assay: dHvH prot_conc:18.6 microM ; Experimental Assay: dG buffers:Phosphate: 10 mM, prot_conc:18.6 microM ; Experimental Assay: dCp buffers:Phosphate: 10 mM, prot_conc:9.1 microM ; Experimental Assay: dHvH prot_conc:9.1 microM ; Experimental Assay: dG buffers:Phosphate: 10 mM, prot_conc:9.1 microM ; Experimental Assay: dCp buffers:Phosphate: 10 mM, prot_conc:4.2 microM ; Experimental Assay: dHvH prot_conc:4.2 microM ; Experimental Assay: dG buffers:Phosphate: 10 mM, prot_conc:4.2 microM ; Experimental Assay: dCp prot_conc:265.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:265.0 microM ; Experimental Assay: dG prot_conc:265.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: dCp prot_conc:237.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:237.1 microM ; Experimental Assay: dG prot_conc:237.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: dCp prot_conc:229.2 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:229.2 microM ; Experimental Assay: dG prot_conc:229.2 microM, buffers:HEPES: 10 mM ; Experimental Assay: dCp prot_conc:115.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:115.1 microM ; Experimental Assay: dG prot_conc:115.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: dCp prot_conc:80.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:80.0 microM ; Experimental Assay: dG prot_conc:80.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: dCp prot_conc:71.4 microM, buffers:HEPES: 10 mM ; Experimental Assay: dHcal prot_conc:71.4 microM ; Experimental Assay: dG prot_conc:71.4 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm buffers:Phosphate: 10 mM, prot_conc:34.7 microM ; Experimental Assay: Tm buffers:Phosphate: 10 mM, prot_conc:25.5 microM ; Experimental Assay: Tm buffers:Phosphate: 10 mM, prot_conc:18.6 microM ; Experimental Assay: Tm buffers:Phosphate: 10 mM, prot_conc:9.1 microM ; Experimental Assay: Tm buffers:Phosphate: 10 mM, prot_conc:4.2 microM ; Experimental Assay: Tm prot_conc:265.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm prot_conc:237.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm prot_conc:229.2 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm prot_conc:115.1 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm prot_conc:80.0 microM, buffers:HEPES: 10 mM ; Experimental Assay: Tm prot_conc:71.4 microM, buffers:HEPES: 10 mM
Libraries Mutations for sequence A:KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILM/B:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/C:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/D:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/E:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/F:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/G:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/H:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/I:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/J:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/K:TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR/L:KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILM

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
4ULL 1997-04-01 SOLUTION NMR STRUCTURE OF VEROTOXIN-1 B-SUBUNIT FROM E. COLI, 5 STRUCTURES
1C4Q 2000-09-20 1.52 MUTATED SHIGA-LIKE TOXIN B SUBUNIT (F30A/W34A) COMPLEXED WITH RECEPTOR GB3 ANALOGUE
1C48 2000-08-16 1.6 MUTATED SHIGA-LIKE TOXIN B SUBUNIT (G62T)
1D1I 2000-09-20 1.7 MUTATED SHIGA-LIKE TOXIN B SUBUNIT (W34A) COMPLEXED WITH RECEPTOR GB3 ANALOGUE
1D1K 2000-09-20 2.0 MUTATED SHIGA-LIKE TOXIN B SUBUNIT (D17E/W34A) COMPLEXED WITH RECEPTOR GB3 ANALOGUE
2XSC 2010-10-13 2.05 Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli
1CQF 2000-08-07 2.2 THE COMPLEX OF THE MUTATED SHIGA TOXIN B SUBUNIT AND GB3 TRISACCHARIDE
1DM0 1999-12-30 2.5 SHIGA TOXIN
1R4Q 2004-05-11 2.5 Shiga toxin
1CZW 2000-09-08 2.5 STRUCTURE OF THE W34A MUTANT OF SHIGA-LIKE TOXIN I B SUBUNIT
1CZG 2000-09-13 2.5 STRUCTURE OF THE G62T MUTANT OF SHIGA-LIKE TOXIN I B SUBUNIT
1BOS 1999-02-02 2.8 SHIGA-LIKE TOXIN COMPLEXED WITH ITS RECEPTOR
2C5C 2006-11-27 2.94 Shiga-like toxin 1 B subunit complexed with a bivalent inhibitor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
199.4 A,L Shiga toxin subunit A P08026 STXA_BPH19
200.0 A,L Shiga toxin subunit A Q9FBI2 STXA_SHIDY
200.0 A,L Shiga toxin subunit A Q32GM1 STXA_SHIDS
200.0 A,L Shiga toxin subunit A P10149 STXA_BPH30
200.0 A,L Shiga toxin subunit A Q779K4 STXA_BP788
1000.0 B,C,D,E,F,G,H,I,J,K Shiga toxin subunit A Q7BQ98 STXB_SHIDY
1000.0 B,C,D,E,F,G,H,I,J,K Shiga toxin subunit A Q32GM0 STXB_SHIDS
1000.0 B,C,D,E,F,G,H,I,J,K Shiga toxin subunit A P69178 STXB_BPH30
1000.0 B,C,D,E,F,G,H,I,J,K Shiga toxin subunit A P69179 STXB_BPH19
1000.0 B,C,D,E,F,G,H,I,J,K Shiga toxin subunit A Q779K3 STXB_BP788