Sites of allosteric shift in the structure of the cyclic AMP receptor protein

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.     

Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets

This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed.     

Investigation of the cAMP receptor protein secondary structure by Raman spectroscopy

Raman spectroscopy was employed to examine the secondary structure of the cAMP receptor protein (CRP). Spectra were obtained over the range 400-1900 cm-1 from solutions of CRP and from CRP-cAMP cocrystals. The spectra of CRP dissolved in 30 mM sodium phosphate and 0.15 M NaCl buffered at either pH 6 or pH 8 or dissolved in 0.15-0.2 M NaCl at protein concentrations of 5, 15, and 30 mg/mL were examined. Estimates of the secondary structure distribution were made by analyzing the amide I region of the spectra (1630-1700 cm-1). CRP secondary structure distributions were essentially the same in either pH and at all protein concentrations examined. The amide I analyses indicated a structural distribution of 44% alpha-helix, 28% beta-strand, 18% turn, and 10% undefined for CRP in solution. Raman spectra of CRP-cAMP cocrystals differed from the spectra of CRP in solution. Some differences were assigned to interfering background bands, whereas other spectral differences were attributed to changes in CRP structure. Differences in the amide III region and in the intensity at 935 cm-1 were consistent with alterations in secondary structure. Analysis of the amide I region of the CRP-cAMP cocrystal spectrum indicated a secondary structure distribution of 37% alpha-helix, 33% beta-strand, 17% turn, and 12% undefined. This result is in agreement with a published secondary structure distribution derived from X-ray analysis of CRP-cAMP cocrystals (37% alpha-helix and 36% beta-strand).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.

Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP受体蛋白在细菌中的转录调控作用研究进展

细菌中cAMP受体蛋白（CRP）对依赖其的启动子的转录起始调控机制及其他调控作用已经得到详细深入的研究。CRP由cAMP激活,并与之结合形成CRP-cAMP复合体,后者与启动子上的特异位点结合,然后与RNA聚合酶相互作用,增强其与启动子的结合能力,从而起始转录。CRP-cAMP复合体与启动子不同的结合方式决定了CRP-9RNA聚合酶之间存在多种不同的作用方式。除了在碳源代谢方面的重要调控作用,CRP对细菌其他代谢途径也有调控作用。     

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.     

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket.

The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.     

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.Abbreviations CRP cyclic AMP receptor protein - NATA N-acetyltryptophanamide - FQRS fluorescence-quenching-resolved spectra - FDCD fluorescence-detected circular dichroism - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - FPLC fast protein liquid chromatography     

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.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

Interaction of CRP L124 with cAMP affects CRP cAMP binding constants,cAMP binding cooperativity,and CRP allostery

A cyclic nucleotide-binding pocket of the CRP dimer is composed of amino acid residues contributed by both subunits. Leucine (L) 124 of one subunit packs against the adenine ring of cAMP bound to the opposing subunit. We have undertaken a study designed to evaluate the role of L124 in CRP allostery. Wild-type (WT) apo-CRP is a 47 kDa protease-resistant dimer composed of identical subunits that exhibits a biphasic isotherm in cAMP titration studies. The WT CRP-cAMP complex is a protease-sensitive dimer degraded by protease to a dimer core that ranges between 26.5 and 30.5 kDa. Substitution of L124 with isoleucine (I), valine (V), cysteine (C), or alanine (A) generated a series of CRP variants that exhibited unique differences in apo-CRP resistance to protease, the mass of the core fragments generated in protease digestion reactions, cAMP-mediated allostery, and CRP-cAMP complex functionality. Differences in the affinity of the position 124 CRP variants for cAMP were observed. The binding constants that drive the formation of the WT and L124I CRP-cAMP complexes deviated by not more than a factor of 1.5. In contrast, the L124V, L124A, and L124C forms of CRP exhibited both a decreased K(cAMP1)(app) and an increased K(cAMP2)(app) to produce 2.4-, 55-, and 204-fold reductions, respectively, in the difference between these two parameters compared to that observed for WT CRP. The data indicate that the van der Waals volume and/or the hyrophobicity of the L124 side chain are important determinants of CRP cAMP binding properties and affect, either directly or indirectly, cAMP-mediated conformation changes in CRP.