Kinetic studies of cAMP-induced allosteric changes in mutants T127I, S128A, and T127I/S128A of the cAMP receptor protein from Escherichia coli.
The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys178, localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP.cAMP2) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: kc = 3.62 s-1 and k-c = 3.13s-1 for CRP T127I and kc = 0.42 s-1 and k-c = 0.78 s-1 for CRP S128A. These values can be compared with kc = 9.7 s-1 and k-c = 0.31 s-1 for wild-type CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP.cAMP4 complex results from displacement of equilibrium between the two forms of the CRP.cAMP2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser128 and Thr127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP.
Polit
Agnieszka
A
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
Bonarek
Piotr
P
Kepys
Barbara
B
Kedracka-Krok
Sylwia
S
Górecki
Andrzej
A
Wasylewski
Zygmunt
Z
D000495:Allosteric Site; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013451:Sulfonic Acids / Q000494:pharmacology; D013816:Thermodynamics; D013997:Time Factors
C418282:N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid; D009281:Naphthalenes; D013451:Sulfonic Acids; D000242:Cyclic AMP