An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.


Abstract

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain. Study holds ProTherm entries: 3846, 3847, 3848, 3849 Extra Details: computer model building; DNA binding activity; cysteine;,stability

Submission Details

ID: 8AgrQwzr3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Sauer RT;Hehir K;Stearman RS;Weiss MA;Jeitler-Nilsson A;Suchanek EG;Pabo CO,Biochemistry (1986) An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor. PMID:3539184
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
1F39 2000-06-01T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR C-TERMINAL DOMAIN
1KCA 2001-11-07T00:00:00+0000 2.91 Crystal Structure of the lambda Repressor C-terminal Domain Octamer
1LLI 1994-03-25T00:00:00+0000 2.1 THE CRYSTAL STRUCTURE OF A MUTANT PROTEIN WITH ALTERED BUT IMPROVED HYDROPHOBIC CORE PACKING
1LMB 1991-11-05T00:00:00+0000 1.8 REFINED 1.8 ANGSTROM CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR-OPERATOR COMPLEX
1LRP 1987-12-04T00:00:00+0000 3.2 COMPARISON OF THE STRUCTURES OF CRO AND LAMBDA REPRESSOR PROTEINS FROM BACTERIOPHAGE LAMBDA
1RIO 2003-11-17T00:00:00+0000 2.3 Structure of bacteriophage lambda cI-NTD in complex with sigma-region4 of Thermus aquaticus bound to DNA
3BDN 2007-11-15T00:00:00+0000 3.91 Crystal Structure of the Lambda Repressor
3KZ3 2009-12-07T00:00:00+0000 1.64 A structure of a lambda repressor fragment mutant
3WOA 2013-12-25T00:00:00+0000 2.0 Crystal structure of lambda repressor (1-45) fused with maltose-binding protein
5ZCA 2018-02-16T00:00:00+0000 1.8 Crystal structure of lambda repressor (1-20) fused with maltose-binding protein

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Repressor protein cI P03034 RPC1_LAMBD