Interdomain interaction of cyclic AMP receptor protein in the absence of cyclic AMP

Interdomain interaction of apo-cyclic AMP receptor protein (apo-CRP) was qualified using its isolated domains. The cAMP-binding domain was prepared by a limited proteolysis, while the DNA-binding domain was constructed as a recombinant protein. Three different regions making interdomain contacts in apo-CRP were identified by a sequence-specific comparison of the HSQC spectra. The results indicated that apo-CRP possesses characteristic modules of interdomain interaction that are properly organized to suppress activity and to sense and transfer the cAMP binding signals. Particularly, the inertness of the DNA-binding motif in apo-CRP was attributable to the participation of F-helices in the interdomain contacts.     

Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.

Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.     

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.     

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.     

Near ultraviolet circular dichroism study of the cyclic AMP receptor protein, its NH2-terminal domain and their interaction with cyclic AMP.

Circular dichroism in the near ultraviolet wavelength range was employed to examine conformational features of CRP (a dimer with a chain of 209 amino acids) and of its subtilisin core -alpha CRP- which retains the cAMP binding site (a dimer spanning the sequence 1-117). Binding of the ligand cAMP (allosteric activator), as well as cGMP was also investigated. The well resolved transitions could be assigned to the various classes of aromatic amino acid residues in the two proteins. In addition to signals which are attributable to the missing aromatic residues (Phe-136 and Tyr-206) the difference spectrum (CRP minus alpha CRP) shows a significant perturbation of a tryptophanyl contribution centred at 296 nm. From the available X-ray structure of the cAMP-CRP complex we are led to conclude that a conformational reorganisation takes place in the alpha CRP. A very large negative maximum is observed at 255 nm when cAMP binds to CRP and to alpha CRP. The maximum effect is observed in both cases at a ratio of one ligand bound per protomer. In the 280-300 nm wavelength range a smaller but significant perturbation affects specifically the spectra and reveals different cAMP-induced conformational changes in the two proteins. We propose that the major (255 nm) contribution to the perturbation spectrum of bound cAMP, and the qualitatively similar signal for cGMP, reflects an immobilisation of the sugar and adenine moieties of the bound ligand in an almost anti-conformation for both CRP and alpha CRP.     

Structural understanding of the allosteric conformational change of cyclic AMP receptor protein by cyclic AMP binding

Cyclic AMP receptor protein (CRP) plays a key role in the regulation of more than 150 genes. CRP is allosterically activated by cyclic AMP and binds to specific DNA sites. A structural understanding of this allosteric conformational change, which is essential for its function, is still lacking because the structure of apo-CRP has not been solved. Therefore, we performed various NMR experiments to obtain apo-CRP structural data. The secondary structure of apo-CRP was determined by analyses of the NOE connectivities, the amide proton exchange rates, and the (1)H-(15)N steady-state NOE values. A combination of the CSI-method and TALOS prediction was also used to supplement the determination of the secondary structure of apo-CRP. This secondary structure of apo-CRP was compared with the known structure of cyclic AMP-bound CRP. The results suggest that the allosteric conformational change of CRP caused by cyclic AMP binding involves subunit realignment and domain rearrangement, resulting in the exposure of helix F onto the surface of the protein. Additionally, the results of the one-dimensional [(13)C]carbonyl NMR experiments show that the conformational change of CRP caused by the binding of cyclic GMP, an analogue of cyclic AMP, is different from that caused by cyclic AMP binding.     

Regulation of acetylcholine receptor by cyclic AMP

In primary cultures of chick 11-day embryonic tissue a number of phosphodiesterase inhibitors were found to elevate acetylcholine receptor levels. Of these agents, Ro20-1724 was the most effective, elevating surface receptor content by 2-fold after 48 h of treatment. 8-Br-cAMP and cholera toxin, a natural activator of adenylate cyclase, mimicked the effect of Ro20-1724, while 8-Br-cGMP and dibutyryl cGMP had no effect. Cholera toxin, 8-Br-cAMP, and Ro20-1724 all increased the insertion rate of new receptor into the surface membrane without altering degradation. The enhanced insertion appears related to an actual increase in synthesis since total acetylcholine receptor was elevated by exposure to cholera toxin. In contrast, no change in creatine phosphokinase activity, myosin heavy chain content, or [35S] methionine incorporation into total cellular protein was observed during cholera toxin treatment. These results suggest that cAMP plays a role in the regulation of acetylcholine receptor.     

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.     

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.     

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.