title: Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.
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.
authors.affiliation: From the Departments of Biology.
authors.forename: Sanjiva M
authors.initials: SM
authors.lastname: Gunasekara
authors.affiliation: From the Departments of Biology.
authors.forename: Matt N
authors.initials: MN
authors.lastname: Hicks
authors.affiliation: Computer Science, and.
authors.forename: Jin
authors.initials: J
authors.lastname: Park
authors.affiliation: Chemistry, California State University Fresno, Fresno, California 93740.
authors.forename: Cory L
authors.initials: CL
authors.lastname: Brooks
authors.affiliation: the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, and.
authors.forename: Jose
authors.initials: J
authors.lastname: Serate
authors.affiliation: From the Departments of Biology.
authors.forename: Cameron V
authors.initials: CV
authors.lastname: Saunders
authors.affiliation: From the Departments of Biology.
authors.forename: Simranjeet K
authors.initials: SK
authors.lastname: Grover
authors.affiliation: Chemistry, California State University Fresno, Fresno, California 93740.
authors.forename: Joy J
authors.initials: JJ
authors.lastname: Goto
authors.affiliation: the Department of Life Science and Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Jin-Won
authors.initials: JW
authors.lastname: Lee
authors.affiliation: From the Departments of Biology, hyoun@csufresno.edu.
authors.forename: Hwan
authors.initials: H
authors.lastname: Youn
mesh_terms: D000494:Allosteric Regulation; D003062:Codon; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D003062:Codon; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C481662:crp protein, E coli; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP
keywords: CRP homolog; DNA binding; Escherichia coli (E. coli); allosteric regulation; cAMP pocket; cAMP receptor protein; cyclic AMP (cAMP); protein evolution; transcription factor