A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.     

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.     

Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein

Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.     

Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.     

Structure and dynamics of the modular halves of Escherichia coli cyclic AMP receptor protein

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains. To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides. The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets. The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices. The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209. The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP. Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP. In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific. Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding. The acquisition of this unique property is intimately associated with the dynamics of the molecule.     

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.     

In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro. This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA. No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative. Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound. Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding. We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer. CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.     

Cyclic AMP-mediated intersubunit disulfide crosslinking of the cyclic AMP receptor protein of Escherichia coli.

The protomeric form of the cyclic AMP receptor protein (CRP) of Escherichia coli is composed of two identical subunits of molecular weight 22,500 and contains two buried and two available cysteine residues. Titration of the two available cysteines with DTNB⁴ eliminates cyclic AMP-dependent DNA binding activity which is regenerated by incubating the modified protein with β-mercaptoethanol. In the absence of cAMP, the formation of the TNB anion from DTNB and the incorporation of [¹⁴C]TNB into CRP are approximately stoichiometric. In the presence of cAMP, there is an increase in the rate of formation of the TNB anion while the incorporation of [¹⁴C]TNB into CRP is markedly inhibited. These observations are reconciled by the observation that cAMP induces DTNB-mediated disulfide crosslinking of the two available sulfhydryls to produce a species migrating as a 45,000 molecular weight subunit on SDS-polyacrylamide gels. A mechanism is suggested by which an intersubunit, intraprotomer disulfide bond is produced by secondary disulfide interchange after the incorporation of the initial TNB group. Based on the observation of cAMP-mediated disulfide crosslinking, the available cysteines of the DNA binding region are proposed to reside in close proximity as part of an antiparallel β-sheet structure formed by the two carboxyl proximal polypeptides when CRP is in the DNA binding conformation.     

Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.