Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.


Abstract

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 7271, 7272 Extra Details: conformational free energy; hydrogen bond; NMR;,protein stability; protein structure

Submission Details

ID: SfeDNz7y3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Hammen PK;Scholtz JM;Anderson JW;Waygood EB;Klevit RE,Protein Sci. (1995) Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy. PMID:7663349
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
1GGR 2000-11-15 COMPLEX OF ENZYME IIAGLC AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
3EZB 1999-12-16 COMPLEX OF THE AMINO TERMINAL DOMAIN OF ENZYME I AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI
2LRK 2012-05-16 Solution Structures of the IIA(Chitobiose)-HPr complex of the N,N'-Diacetylchitobiose
1PFH 1995-11-14 THE PHOSPHORYLATED FORM OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR
1J6T 2002-11-13 COMPLEX OF ENZYME IIAMTL AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
1HDN 1994-06-22 THE HIGH-RESOLUTION STRUCTURE OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI DETERMINED BY RESTRAINED MOLECULAR DYNAMICS FROM NMR NUCLEAR OVERHAUSER EFFECT DATA
2LRL 2012-05-16 Solution Structures of the IIA(Chitobiose)-HPr complex of the N,N'-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System
1VRC 2005-04-19 Complex of enzyme IIAmannose and the histidine-containing phosphocarrier protein HPr from escherichia coli nmr, restrained regularized mean structure
2XDF 2010-09-22 Solution Structure of the Enzyme I Dimer Complexed with HPr Using Residual Dipolar Couplings and Small Angle X-Ray Scattering
3EZE 1998-12-16 COMPLEX OF THE AMINO TERMINAL DOMAIN OF ENZYME I AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
3EZA 1999-05-25 COMPLEX OF THE AMINO TERMINAL DOMAIN OF ENZYME I AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
3CCD 2008-10-21 1.0 1.0 A Structure of Post-Succinimide His15Asp HPr
1OPD 1997-08-20 1.5 HISTIDINE-CONTAINING PROTEIN (HPR), MUTANT WITH SER 46 REPLACED BY ASP (S46D)
1CM3 2000-05-17 1.6 HIS15ASP HPR FROM E. COLI
1CM2 2000-05-17 1.8 STRUCTURE OF HIS15ASP HPR AFTER HYDROLYSIS OF RINGED SPECIES.
1POH 1994-01-31 2.0 THE 2.0 ANGSTROMS RESOLUTION STRUCTURE OF ESCHERICHIA COLI HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR: A REDETERMINATION
4XWJ 2015-10-21 2.1 Histidine-containing phosphocarrier protein (HPr) and antisigma factor Rsd complex
2JEL 1998-05-27 2.5 JEL42 FAB/HPR COMPLEX
5YA1 2018-07-11 2.7 crystal structure of LsrK-HPr complex with ATP
5YA2 2018-07-11 2.7 Crystal structure of LsrK-HPr complex with ADP
5YA0 2018-07-11 3.0 Crystal structure of LsrK and HPr complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.8 Phosphocarrier protein HPr P16481 PTHP_KLEPN
100.0 Phosphocarrier protein HPr P0AA09 PTHP_SHIFL
100.0 Phosphocarrier protein HPr P0AA07 PTHP_SALTY
100.0 Phosphocarrier protein HPr P0AA08 PTHP_SALTI
100.0 Phosphocarrier protein HPr P0AA04 PTHP_ECOLI
100.0 Phosphocarrier protein HPr P0AA05 PTHP_ECOL6
100.0 Phosphocarrier protein HPr P0AA06 PTHP_ECO57