Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr127 and Ser128, are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformati...

Full description

Saved in:

Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 290; no. 44; pp. 26587 - 26596
Main Authors:	Gunasekara, Sanjiva M., Hicks, Matt N., Park, Jin, Brooks, Cory L., Serate, Jose, Saunders, Cameron V., Grover, Simranjeet K., Goto, Joy J., Lee, Jin-Won, Youn, Hwan
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 30-10-2015 American Society for Biochemistry and Molecular Biology
Subjects:	Allosteric Regulation cAMP pocket cAMP receptor protein Codon CRP homolog Crystallography, X-Ray cyclic AMP (cAMP) Cyclic AMP - chemistry Cyclic AMP - metabolism Cyclic AMP Receptor Protein - chemistry Cyclic AMP Receptor Protein - genetics Cyclic AMP Receptor Protein - metabolism Directed Molecular Evolution DNA binding DNA, Bacterial - chemistry DNA, Bacterial - metabolism Escherichia coli (E. coli) Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Evolution, Molecular Gene Expression Regulation, Bacterial Hydrogen Bonding Models, Molecular Mutagenesis, Site-Directed Protein Binding Protein Conformation protein evolution Protein Structure and Folding Serine - chemistry Serine - metabolism Threonine - chemistry Threonine - metabolism transcription factor Transcription, Genetic transcription factor CRP homolog protein evolution Escherichia coli (E. coli) cyclic AMP (cAMP) allosteric regulation DNA binding cAMP pocket cAMP receptor protein
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr127 and Ser128, are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr127 and Ser128 residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr127 and Ser128 was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP. Background: Thr127 and Ser128 are important for the function of the Escherichia coli CRP (cAMP receptor protein). Results: Thr/Ser, Thr/Thr, and Thr/Asn pairs at the positions are optimal for CRP function. Conclusion: Evolutionarily conserved residue pairs at the positions provide high cAMP affinity while keeping CRP inactive without cAMP. Significance: There are multiple evolutionary strategies for cAMP sensing in CRP.
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.M115.678474