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

Title
Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket
Author
이진원
Keywords
CATABOLITE ACTIVATOR PROTEIN; VIRULENCE FACTOR REGULATOR; CYCLIC-AMP; TRANSCRIPTION ACTIVATION; PSEUDOMONAS-AERUGINOSA; CONFORMATIONAL-CHANGES; CRYSTAL-STRUCTURE; DNA-BINDING; SUGGESTS; CAP
Issue Date
2015-10
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Citation
JOURNAL OF BIOLOGICAL CHEMISTRY, v. 290, NO 44, Page. 26587-26596
Abstract
The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), 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 Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) 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.
URI
http://www.jbc.org/content/290/44/26587.shorthttp://hdl.handle.net/20.500.11754/27952
ISSN
0021-9258; 1083-351X
DOI
10.1074/jbc.M115.678474
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > LIFE SCIENCE(생명과학과) > Articles
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