Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8.


Abstract

Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the stability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA. Study holds ProTherm entries: 889, 890, 891, 892, 893, 894, 13448 Extra Details: additive : EDTA(0.2 mM), Arc repressor; DNA binding affinity; hydrogen bond;,kinetics; stability

Submission Details

ID: Qx3Lzt9K

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Schildbach JF;Milla ME;Jeffrey PD;Raumann BE;Sauer RT,Biochemistry (1995) Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8. PMID:7827088
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
1NLA 2003-03-18 Solution Structure of Switch Arc, a Mutant with 3(10) Helices Replacing a Wild-Type Beta-Ribbon
1ARR 1994-01-31 RELAXATION MATRIX REFINEMENT OF THE SOLUTION STRUCTURE OF THE ARC REPRESSOR
1QTG 1999-07-12 AVERAGED NMR MODEL OF SWITCH ARC, A DOUBLE MUTANT OF ARC REPRESSOR
1ARQ 1994-01-31 RELAXATION MATRIX REFINEMENT OF THE SOLUTION STRUCTURE OF THE ARC REPRESSOR
1B28 1999-11-03 ARC REPRESSOR MYL MUTANT FROM SALMONELLA BACTERIOPHAGE P22
1BAZ 1998-06-17 1.9 ARC REPRESSOR MUTANT PHE10VAL
1U9P 2005-02-15 1.9 Permuted single-chain Arc
1MYL 1995-01-26 2.4 SUBSTITUTING HYDROPHOBIC RESIDUES FOR A BURIED SALT BRIDGE ENHANCES PROTEIN STABILITY BUT DOES NOT REDUCE CONFORMATIONAL SPECIFICITY
1MYK 1995-01-26 2.4 CRYSTAL STRUCTURE, FOLDING, AND OPERATOR BINDING OF THE HYPERSTABLE ARC REPRESSOR MUTANT PL8
1BDT 1999-02-16 2.5 WILD TYPE GENE-REGULATING PROTEIN ARC/DNA COMPLEX
1PAR 1994-07-31 2.6 DNA RECOGNITION BY BETA-SHEETS IN THE ARC REPRESSOR-OPERATOR CRYSTAL STRUCTURE
1BDV 1999-01-06 2.8 ARC FV10 COCRYSTAL

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Transcriptional repressor arc P03050 RARC_BPP22