Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

Allosteric control of cAMP receptor binding dynamics

The intrinsic fluorescence of the cyclic AMP receptor is a sensitive indicator of the reaction with DNA, but signals are perturbed by a photoreaction. A ratio procedure is shown to be useful for correction. The reaction of the protein with DNA indicated by corrected transients extends over a broad time range not only at low salt concentrations but also at physiological salt concentrations. The initial binding step can be recorded preferentially at low salt pH 7 and is shown to be very similar for specific and nonspecific DNA. The rate constant for initial binding at 13.5 mM salt pH 7 is 2 × 10(8) M(-1) s(-1). Slow reaction steps up to times of several hundred seconds are observed both at low and high salt; the magnitude and sign of fluorescence amplitudes are strongly dependent on salt and pH. At 100 mM salt pH 8, the slow reaction step observed for the binding of the cyclic AMP receptor protein to promoter DNA is strongly shifted to longer times upon reduction of the cAMP concentration. The observed cAMP dependence is described quantitatively by a model implying that binding of the receptor to promoter DNA requires two cAMP molecules per protein dimer and is not consistent with a model assuming that a single cAMP is sufficient for activation. The rate constant for binding of the protein·dimer·(cAMP)(2) complex to the promoter is 1.3 × 10(8) M(-1) s(-1), close to the limit of diffusion control. Equilibration of specific complexes takes ~100 s at physiological concentrations of the reaction components.     

The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding

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 A. 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 anti-cAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr(127) and Ser(128) residues in the C alpha-helix of wild type CRP. This replacement induces flexibility in the C alpha-helix at Ala(128), which swings the C-terminal domain of the open subunit more toward the N-terminal domain in the T127L/S128A double mutant of CRP (CRP*) than is observed in the open subunit of cAMP-ligated CRP. Isothermal titration calorimetry measurements on the binding of cAMP to CRP* show that the binding mechanism changes from an exothermic independent two-site binding mechanism at pH 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar to that observed for CRP at both pH levels. Differential scanning calorimetry measurements exhibit a broadening of the thermal denaturation transition of CRP* relative to that of CRP at pH 7.0 but similar to the multipeak transitions observed for cAMP-ligated CRP. These properties and the bound syn-cAMP ligand, which has only been previously observed in the DNA bound x-ray crystal structure of cAMP-ligated CRP by Passner and Steitz (Passner, J. M., and Steitz, T. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2843-2847), imply that the cAMP-ligated CRP* structure is closer to the conformation of the allosterically activated structure than cAMP-ligated CRP. This may be induced by the unique flexibility at Ala(128) and/or by the bound syn-cAMP in the hinge region of CRP*.     

Site-directed polyclonal antibodies inhibit binding of activated estrogen receptor to DNA

We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.     

Persistent cAMP binding by the chemotactic cAMP receptor in the presence of a detergent in Dictyostelium discoideum

We have investigated the effect of a number of detergents on the chemotactic cAMP receptor of Dictyostelium discoideum. 13 detergents were tested; cAMP binding was well preserved only in the presence CHAPS (3[3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) and Zwittergent 3–8 (N-octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate). In the presence of Zwittergent 3–8, cAMP bound to the receptor rapidly exchanged with free cAMP. In contrast, cAMP was persistently bound to the receptor following the addition of CHAPS to membrane-bound receptors pre-equilibrated with cAMP. Binding isotherms indicated that all cAMP-binding sites were similarly affected by CHAPS. The cyclic nucleotide binding specificity of the binding sites that became persistently occupied by cAMP was identical to that of the chemotactic cAMP receptor. Cyclic AMP was not chemically modified by persistent binding. The non-exchanging cAMP-receptor complex was insensitive to modulation by guanine nucleotides and salts such as CaCl₂, MgCl₂, potassium phosphate and ammonium sulphate. We conclude that CHAPS freezes the cAMP-receptor, blocking exchange of free ligand with empty or occupied cAMP-binding sites.     

噬菌体吸附及受体结合蛋白(RBP)的研究进展与应用

噬菌体通过受体结合蛋白(Receptor binding protein,RBP)结合到细菌表面,其过程需要复杂的原子结构的参与和构象改变.针对噬菌体侵染,细菌发展了多种抗性机制,同时,噬菌体也进化出多种逃逸宿主抗性的机制.对噬菌体与细菌间"吸附-抗吸附-逃逸过程"的探索有助于我们理解噬菌体与细菌共进化的过程,对科学发...     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

Identification and characterization of the cAMP binding proteins of yeast by photoaffinity labeling.

Modulation of a membrane glycoprotein, approximate molecular weight 200,000, in concert with active ionic flux has been shown in a human neuroblastoma cell line. The modulating agent was 2% dimethyl sulfoxide. Other neuronal properties, acetylcholinesterase and choline acetyltransferase, were also modulated but to a lesser extent. The appearance of this glycoprotein on the surface of both human and mouse neuroblastoma cells only under conditions of differentiation leads to the suggestion that it is directly involved with the active Na⁺ channels.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.