Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

Autor:	Gunasekara SM; From the Departments of Biology., Hicks MN; From the Departments of Biology., Park J; Computer Science, and., Brooks CL; Chemistry, California State University Fresno, Fresno, California 93740., Serate J; the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, and., Saunders CV; From the Departments of Biology., Grover SK; From the Departments of Biology., Goto JJ; Chemistry, California State University Fresno, Fresno, California 93740., Lee JW; the Department of Life Science and Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea., Youn H; From the Departments of Biology, hyoun@csufresno.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2015 Oct 30; Vol. 290 (44), pp. 26587-96. Date of Electronic Publication: 2015 Sep 16.
DOI:	10.1074/jbc.M115.678474
Abstrakt:	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. (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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