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)     

Sensitization of the Escherichia coli cyclic AMP receptor protein to trypsin cleavage by polydeoxyribonucleotides and polyribonucleotides

In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N-terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP.     

Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.     

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.     

Cross-linking of the cAMP receptor protein of Escherichia coli by o-phenylenedimaleimide as a probe of conformation.

Reaction of the cAMP (cyclic adenosine 3'--5'-monophosphate) receptor protein (CRP) of Escherichia coli with the bifunctional reagent o-phenylenedimaleimide (oPDM) results in the cross-linking of the two subunits of a CRP protomer. In the presence of cAMP the rate of cross-linking increases. CRP modified with oPDM retains [3H]cAMP binding activity but loses [3H]d(I-C)n binding activity. Proteolysis of cross-linked CRP gives distinct sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns depending upon whether cAMP was present during the reaction with oPDM. CRP cross-linked in the absence of cAMP retains the same relative resistance to proteolysis as unmodified CRP. The presence of 0.1 mM cAMP during proteolysis results in the production of two fragments, one of approximately 13 000 daltons and a second of approximately 20 000 daltons. CRP cross-linked with oPDM in the presence of cAMP (then dialyzed to remove cAMP) remains sensitive to alpha-chymotrypsin digestion even in the absence of added cAMP producing only the 13 000-dalton fragment. It is suggested that the nature of the oPDM cross-link is a consequence of the conformational state of CRP.     

The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements.

The structure of complexes formed between cAMP receptor protein (CRP) and various restriction fragments from the promoter region of the lactose operon has been analysed by measurements of electrodichroism. Binding of CRP to a 62-bp fragment containing the major site leads to an increase of the rotation time constant from 0.33 to 0.43 microseconds; addition of cAMP to the complex induces a decrease to 0.25 microseconds. Similar data are obtained for a 80-bp fragment containing the operator site; however, in this case the decrease of the rotation time for the specific complex is only observed when the salt concentration is increased from 3 to 13 mM. A 203-bp fragment containing both sites showed a corresponding change after pre-incubation at 50 mM salt. The salt dependence of the rotation time for the specific complex formed with the 203-bp fragment also indicates that a compact structure is formed at 13 mM salt, whereas the structure is not as compact at 3 mM salt. A 98-bp fragment without specific CRP sites did not reveal changes corresponding to those observed for the specific fragments. The rotation time constants together with the dichroism amplitudes indicate that binding of CRP to specific sites in the presence of cAMP leads to the formation of compact structures, which are consistent with bending of DNA helices. The observed strong salt dependence of the structure is apparently due to electrostatic repulsion between adjoining helix segments.     

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Forster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA-CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.     

CRP subunit association and hinge conformation changes in response to cAMP binding: analysis of C-helix cysteine-substituted CRP

We investigated the characteristics of 13 CRP variants having cysteine substituted at positions 113, 115, 116, 117, 118, 120, 122, 124, 126, 127, 129, 130, or 131, positions that span the length of the CRP C alpha-helix. Under reducing conditions, the WT and all Cys-substituted forms of CRP migrated as 23.5 kDa CRP monomer species on SDS-PAGE gels. In the absence of a reductant, 9 of 13 Cys-substituted forms of CRP including the L113C, S117C, M120C, L124C, V126C, T127C, E129C, K130C, and V131C CRP contained protein that migrated as 47 kDa CRP dimer species on SDS-PAGE gels. CNBr digestion of the protein preparations followed by MALDI-TOF MS analysis of the peptide fragments showed these 47 kDa species to be CRP dimers that originated from disulfide bonds formed between positional-pair C alpha-helix Cys residues. The ratio of monomer CRP and disulfide cross-linked CRP within a Cys-substituted CRP preparation was found to be independent of cAMP for Cys-substituted CRP preparations denatured and renatured in the presence of various cAMP concentrations. This finding suggests that there is no large-scale concerted motion (i.e., scissoring) of the CRP subunits in response to cAMP binding. In addition, we have identified three amino acid residues located along the CRP C alpha-helix that play a role in facilitating the conformation transition of the CRP hinge from that characteristic of apo-CRP to that characteristic of the CRP.cAMP complex.     

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.     

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.     

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.     

Kinetic and structural studies of the allosteric conformational changes induced by binding of cAMP to the cAMP receptor protein from Escherichia coli

The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein responsible for interaction with specific sequences of DNA. In this study, the stopped-flow and time-resolved fluorescence lifetime measurements were used to observe the kinetics of the distance changes between the N-terminal and C-terminal domain of CRP induced by binding of cAMP to high-affinity binding sites. In these measurements, we used the constructed CRP heterodimer, which possesses a single Trp85 residue localized at the N-terminal domain of one CRP subunit, and fluorescently labeled by 1,5-I-AEDANS Cys178 localized at the C-terminal domain of the same subunit or at the opposite one. The F?rster resonance energy transfer method has been used to study the distance changes, induced by binding of cAMP, between Trp85 (fluorescence donor) and Cys178-AEDANS (fluorescence acceptor) in the CRP structure. The obtained results show that the allosteric transitions of CRP at micromolar cAMP concentrations follow the sequential binding model, in which binding of cAMP to high-affinity sites causes a 4 A movement of the C-terminal domain toward N-terminal domains of the protein, with kinetics faster than 2 ms, and CRP adopts the "closed" conformation. This fast process is followed by the slower reorientation of both CRP subunits.     

Turn of Promotor DNA by cAMP Receptor Protein Characterized by Bead Model Simulation of Rotational Diffusion

Abstract

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (< 100 bp). The final values reflect the solvated structure with the same ‘solvation layer’ added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix- turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 Å. According to these data the overall bending angle of our longest DNA fragment is approximately 180°, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.     

Turn of promotor DNA by cAMP receptor protein characterized by bead model simulation of rotational diffusion

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (less than 100 bp). The final values reflect the solvated structure with the same 'solvation layer' added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix-turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA, provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 A. According to these data the overall bending angle of our longest DNA fragment is approximately 180 degrees, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

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.