The Structure of the T127L/S128A Mutant of cAMP Receptor Protein Facilitates Promoter Site Binding

Abstract

The x-ray crystal structure of the cAMP-ligated T127L/ S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 Å. Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain. In addition, water-mediated interactions replace the hydrogen bonding interactions between the N 6 of anticAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr 127 and Ser 128 residues in the C ␣-helix of wild type CRP. This replacement induces flexibility in the C ␣-helix at Ala The allosteric protein cAMP receptor protein (CRP) 1 is activated by the binding of cAMP to enhance the transcription of over 25 genes, which code for enzymes involved in carbohydrate metabolism in Escherichia coli. The x-ray crystal structure of cAMP-ligated CRP consists of two 210-amino acid residue subunits, with the N-terminal domain of each subunit containing a central ␤-barrel connected to an ␣/␤ C-terminal domain via an ␣-helix, which also is the primary interface between the two subunits (1). The N-terminal regulatory domain ␤-strands form an eight-stranded ␤-barrel that binds anti-cAMP, and the Cterminal domain contains a helix-turn-helix motif followed by a small ␤-sheet that binds to the promoter site. One subunit (A) has a "closed" form, where the C-terminal domain is swung in toward the ␣-helical interface, and the other subunit (B) has an "open" form, where the C-terminal domain is swung away from the interface (1). In addition to interactions between the CRP binding site and the phosphate ribose of cAMP, Thr 127 at the ␣-helical interface forms a hydrogen bond with the N 6 of the bound cAMP, and Ser 128 forms a hydrogen bond with the bound cAMP in the other subunit (1). The nature of the conformational change to the allosterically activated conformation has not been determined because the x-ray crystal structure of unligated CRP is not known. Small angle neutron scattering measurements on unligated CRP solutions show that the data best fit the simulated scattering data from a minimized energy structure of CRP with both units in the open form (2). Minimum energy calculations starting with the x-ray structure of both anti-cAMP-ligated subunits in the closed form and both subunits in the open form in solution show that the minimum energy conformation of anti-cAMP-ligated CRP is with both subunits in the closed form (3). In NMR measurements, five sets of histidine resonances are observed for CRP alone and in its complexes with cyclic nucleotides, which also implies that both subunits are in the same conformation in solution (4). The x-ray structure of the cAMP-ligated complex of CRP bound to a 30-base pair DNA duplex, which has the same sequence as that of a promoter site, shows that both subunits are in the closed conformation (5, 6). The implication of these results is that the allost...

Kinetic Studies of cAMP-induced Allosteric Changes in Mutants T127I, S128A, and T127I/S128A of the cAMP Receptor Protein from Escherichia coli

“…This suggests that the protein possesses the conformation of the HTH motif, which is able to recognize the specific sequences of the promoter DNA. This suggestion is supported by the observation that a CRP double mutant containing both the T127I and S128A mutations activates transcription in vivo and in vitro in the absence of cyclic nucleotide monophosphate (14). It is known that substitutions T127I (13) and S128A (12) in CRP increase the negative cooperativity between cAMP-binding sites of the protein.…”

“…Because the two forms of the CRP⅐cAMP 2 complex can exist in different conformational states and only one of them (PЈ) can bind cAMP, the observed increase in the K 0 isomerization constant with the amino acid substitutions can result from the shifting of equilibrium from the inactive to active state of CRP, and the magnitude of this shift depends on substitutions. However, a smaller shift in this equilibrium has been observed in the case of the double mutant of CRP, which can activate transcription in the absence of cAMP, and (as it is believed) this mutant possesses the structure appropriate for DNA binding (14).…”

“…In this study, we report a further kinetic description of cAMP-induced conformational changes in CRP based on the use of different mutants (T127I, S128A, and T127I/S128A). It is well known (12)(13)(14) that all of these mutants exhibit altered allosteric activation of CRP both in vitro and in vivo (15). Because Thr 127 and Ser 128 are directly involved in cAMP binding as well as in intersubunit and interdomain interactions in CRP⅐cAMP complexes (4,16), fast kinetic measurements can provide further insight into the allosteric activation of CRP.…”

“…Because Thr 127 forms a hydrogen bond with N-6 of the bound cAMP and Ser 128 forms a hydrogen bond with cAMP in the other subunit (16,4,23), the mutation of these residues can influence both domain-domain interaction as well as the interaction between the two subunits of CRP. The x-ray structure studies of the cAMP-ligated CRP T127I/S128A double mutant have shown that the S128A substitution induces an increase in flexibility in helix C of the protein, which in turn swings the C-terminal domain more toward the N-terminal domain of CRP (14). We suggest that the observed changes in the kinetic rate constants (k c and k Ϫc ) as well as the changes in the equilibrium of conformational transitions (K C ) in the allosteric activation of the CRP T127I and CRP S128A mutants by cAMP result from conformational reorientation of the HTH motif in the C-terminal domain of the protein.…”

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

“…Some point mutations, either within or just outside of these loops, have been reported to significantly affect the function of CRP, e.g. K52N, D53H, and S62F (beside loop 3) and E72A, K82Q, and S83K (beside loop 4) (21,22,(31)(32)(33)(34). Results from protein footprinting and NMR experiments indicate that the regions, including these loops exhibit significant environmental changes upon cAMP binding (18,19).…”