Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects.


Abstract

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI. Study holds ProTherm entries: 9566, 9567, 9568, 9569, 9570 Extra Details: amino terminal domain of enzyme I

Submission Details

ID: P2s2pXAS3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Ginsburg A;Szczepanowski RH;Ruvinov SB;Nosworthy NJ;Sondej M;Umland TC;Peterkofsky A,Protein Sci. (2000) Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects. PMID:10892802
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
1EZA 1997-01-01T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
1EZB 1997-01-01T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI, NMR, 17 STRUCTURES
1EZC 1997-01-01T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI, NMR, 17 STRUCTURES
1EZD 1997-01-01T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI NMR, 16 STRUCTURES
1ZYM 1996-05-21T00:00:00+0000 2.5 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI
2EZA 1997-05-07T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI, NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
2EZB 1997-05-07T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI, NMR, 14 STRUCTURES
2EZC 1997-05-07T00:00:00+0000 0 AMINO TERMINAL DOMAIN OF ENZYME I FROM ESCHERICHIA COLI, NMR, 14 STRUCTURES
2HWG 2006-08-01T00:00:00+0000 2.7 Structure of phosphorylated Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system
2KX9 2010-04-29T00:00:00+0000 0 Solution Structure of the Enzyme I dimer Using Residual Dipolar Couplings and Small Angle X-Ray Scattering

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.2 Phosphoenolpyruvate-protein phosphotransferase P0A249 PT1_SALTY
97.2 Phosphoenolpyruvate-protein phosphotransferase P0A250 PT1_SALTI
100.0 Phosphoenolpyruvate-protein phosphotransferase P08839 PT1_ECOLI