Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein.
The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.
Youn
Hwan
H
Department of Bacteriology, University of Wisconsin, Madison, 53706, USA.
Kerby
Robert L
RL
Conrad
Mary
M
Roberts
Gary P
GP
D002097:C-Reactive Protein / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011953:Receptors, Cyclic AMP / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D011953:Receptors, Cyclic AMP; D013912:Threonine; D012694:Serine; D002097:C-Reactive Protein; D004247:DNA; D008715:Methionine; D000242:Cyclic AMP; D001616:beta-Galactosidase; D007930:Leucine