Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.
Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Heyduk
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Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Heyduk
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T
Lee
J C
JC
D001665:Binding Sites; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013381:Subtilisins / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D000242:Cyclic AMP; D013381:Subtilisins