Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.


Abstract

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding. Study holds ProTherm entries: 12300, 12301, 12302, 12303, 12304, 12305, 12306 Extra Details: beta-barrel; protein engineering; protein folding; electrostatic interactions;,protein-ligand interaction

Submission Details

ID: RdTduu4k

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Zhang J;Liu ZP;Jones TA;Gierasch LM;Sambrook JF,Proteins (1992) Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability. PMID:1377826
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
1CBI 1995-11-14 2.7 APO-CELLULAR RETINOIC ACID BINDING PROTEIN I
2CBR 1999-12-21 2.8 CELLULAR RETINOIC ACID BINDING PROTEIN I IN COMPLEX WITH A RETINOBENZOIC ACID (AM80)
1CBR 1995-01-26 2.9 CRYSTAL STRUCTURE OF CELLULAR RETINOIC-ACID-BINDING PROTEINS I AND II IN COMPLEX WITH ALL-TRANS-RETINOIC ACID AND A SYNTHETIC RETINOID

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.0 Cellular retinoic acid-binding protein 1 Q5R2J5 RABP1_PELSI
95.6 Cellular retinoic acid-binding protein 1 P40220 RABP1_CHICK
99.3 Cellular retinoic acid-binding protein 1 P29762 RABP1_HUMAN
100.0 Cellular retinoic acid-binding protein 1 P62966 RABP1_RAT
100.0 Cellular retinoic acid-binding protein 1 P62965 RABP1_MOUSE
100.0 Cellular retinoic acid-binding protein 1 P62964 RABP1_BOVIN