Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements*

Susan KruegerDagger , Inna Gorshkova, James Brown, Joel Hoskins, Keith H. McKenney, and Frederick P. Schwarz§

From the Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, Rockville, Maryland 20850 and the Dagger  National Institute of Standards and Technology Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha -helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha -helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The binding of 3',5' cyclic adenosine monophosphate (cAMP) to cAMP receptor protein (CRP)1 activates the transcription of over 20 genes that code for the catabolite enzymes involved in carbohydrate metabolism in Escherichia coli. Results from NMR (1, 2), Raman spectroscopy (3), proteolysis studies (4), small angle x-ray scattering measurements (5), and isothermal titration calorimetry (6) indicate that upon binding to cAMP, CRP undergoes a conformational change to a conformation that promotes specific binding to the catabolite operons to activate transcription in the presence of RNA polymerase. The nature of this conformational change is not known, because only the structures of the cAMP-ligated CRP complex and of the DNA-cAMP-ligated CRP complex have been determined from x-ray crystallography studies (7, 8).

CRP exists as a homodimer of 45,000 g mol-1 consisting of a beta -pleated sheet amino-terminal domain, which contains the cAMP binding site and a carboxyl-terminal domain consisting of several alpha -helices that bind to specific DNA sequences. In the crystal phase, the cAMP-ligated CRP dimer is asymmetric where one monomer is in an "open" form in which the alpha -helices are swung out away from the amino-terminal domain and the other monomer is in a "closed" form in which the alpha -helices are swung in close to the amino-terminal domain (7). The amino acid sequence connecting the two domains is, thus, called the "hinge" region. The x-ray crystal structure of the cAMP-ligated CRP dimer complexed with a 30-base pair DNA sequence is exclusively in the closed form (8). Recent energy minimization computations of the two forms in solution show that the lowest energy conformation of the cAMP-ligated CRP dimer is exclusively the closed form in solution (9). The implication is that the asymmetry of the CRP dimer in the crystal lattice is due to crystal packing forces and that the structural change responsible for the activation of transcription is a change from the open to the closed form in solution. This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region (1), and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP (2).

There is, however, evidence that the conformational change may not simply involve a transition from the open to the closed form of CRP upon binding to cAMP in solution. A comparison of the secondary structure of free CRP and cAMP-ligated CRP by Raman spectroscopy (3) shows a structural shift from 44% alpha -helix, 28% beta -strand, 18% turn, and 10% undefined in the free form to 37% alpha -helix, 33% beta -strand, 17% turn, and 12% undefined in the ligated form, implying that the conformational change does not conserve the alpha -helical and beta -strand structure as it would in a shift of the CRP dimer from the exclusively open to the closed form in solution. A conformational change involving alterations in the alpha -helical and beta -strand structure of CRP is also implied by an empirical model of CRP secondary structure based on its sequence. This sequence-based empirical model predicts alpha -helices in unligated CRP in place of the beta -strands in the amino-terminal domain of the x-ray crystallographic structure of the cAMP-ligated CRP complex (10).

In addition to the contradictory evidence concerning the nature of the conformational change in CRP, there is also experimental evidence that a cAMP-induced conformational change is not always necessary for the activation of transcription. For example, in vivo transcription was observed to be activated even in the absence of cAMP by a mutant of CRP where two amino acid residues have been replaced in the amino-terminal domain of CRP, T127L and S128A (CRP*) (11). This implies that CRP* is already in the transcriptionally activated conformation and that cAMP binding is unlikely to initiate a conformational change.

Small angle neutron scattering (SANS) measurements have been employed to determine the conformation and size of proteins in solution (12). Using D2O as the solvent to maximize the difference in neutron scattering between the solvent and the protein, the conformation of the protein can be determined from the dependence of the scattered neutron intensity on the scattering angle and the size of the protein in terms of the radius of gyration (Rg) can be determined from the scattered neutron intensity near zero scattering angle (13). In addition, the SANS data can be compared with scattered neutron intensity curves generated from the energy-minimized crystallographic x-ray structure of the protein to determine in more detail the conformation of the protein in solution.

In this investigation, SANS measurements were performed on solutions of CRP in the absence and presence of cAMP and compared with model scattered intensity curves calculated from the energy-minimized x-ray structures of CRP dimer in exclusively the closed and in exclusively the open forms. For comparison, SANS measurements were also performed on solutions of CRP* in the absence and presence of cAMP.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The characterization and mutagenesis of the wild type and mutant of CRP have been described previously by Moore (11). A New Brunswick Fermentor Scientific, Inc. SF116 was also used in place of the 5-liter shaker flasks for preparations of CRP and CRP*. An Amersham Pharmacia Biotech Phast SDS-electrophoresis gel with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.2 mass % bromphenol blue and 20 volume % glycerol was run to determine the purity of the protein preparations. A protein analysis was also performed by comparing the absorbance at 562 nm of samples of the solution to those of bovine serum albumin solutions after adding 4 mass % CuSO4 and bicinchoninic acid reagent from Pierce. The protein solutions were dialyzed at 4 °C in 50 mM potassium phosphate-potassium hydroxide buffer (pH = 7.0) containing 0.2 mM sodium EDTA, 0.2 mM dithiothreitol, 0.5 mM KCl, and 5 volume % glycerol (phosphate buffer) in D2O with two changes in the buffer solution. The concentrations of the CRP and the mutant were determined by UV absorption spectroscopy using an extinction coefficient of 3.5 × 104 cm liter mol-1 at 280 nm (14). The ligand solutions were made up by dissolving known masses of the sodium salt of the cyclic nucleotide in the D2O buffer. The sodium salt of cAMP, potassium phosphate, NaOH, EDTA, dithiothreitol, glycerol, Tris, HCl, bromphenol blue, and KCl were reagent grade from Sigma.

SANS Measurements-- The SANS measurements were performed on the NG3 30 m SANS instrument at the National Institute of Standards and Technology Center for Neutron Research in Gaithersburg, MD (15). Unligated CRP solutions were measured at 0.036 and 0.096 mM and unligated CRP* at 0.049 and 0.138 mM in the D2O solvent. CRP was also measured at both concentrations in the presence of 0.270 and 17.1 mM cAMP, whereas CRP* solutions at both concentrations were measured in the presence of 17.1 mM cAMP. To limit aggregation effects, the lower concentration solutions were used to obtain the data at the lower scattering angles where information about the overall size of the protein is obtained, whereas the higher concentration solutions were used to obtain the higher angle data, which provide information about the shape of the protein. In the higher angle scattering region, aggregation effects are much smaller than in the lower angle region, but the scattered intensity is also much lower, so a higher concentration of CRP and CRP* in solution is needed in the higher angle region.

Data were taken at a wavelength lambda  = 5 Å, with a spread Delta lambda /lambda  = 0.34 for the CRP samples and Delta lambda /lambda  = 0.15 for the CRP* samples. A sample-to-detector distance of 5.5 m and a source-to-sample distance of 7.02 m were used for the 0.036 and 0.049 mM samples to obtain the lower angle data. The 0.096 and 0.138 mM samples were measured at a sample-to-detector distance of 1.5 m and a source-to-sample distance of 3.92 m to obtain the higher angle data. The source and sample apertures were 5.0 and 1.27 cm, respectively. Neutrons were detected on a 64.0 × 64.0-cm two-dimensional position-sensitive detector with 1.0-cm resolution. The center of the detector was offset by 20.0 cm at both sample-to-detector distance positions to provide a large overlap between the two configurations and to measure to a large enough scattering angle to allow an accurate determination of the incoherent background scattering due to hydrogen in the sample. The scattered intensity, I, was corrected for solvent absorbance and scattering on a pixel-by-pixel basis using the relation,
I<SUB><UP>cor</UP></SUB>=I<SUB><UP>sample</UP></SUB>−<FENCE>T<SUB><UP>sample</UP></SUB>/T<SUB><UP>solvent</UP></SUB></FENCE>I<SUB><UP>solvent</UP></SUB> (Eq. 1)
where Tsample and Tsolvent are the measured transmissions of neutrons through the sample and solvent, respectively. The corrected data were placed on an absolute scale by calibration against the scattering from a silica gel standard sample and then radially averaged to obtain I(Q) as a function of Q, where Q = 4pi sintheta /lambda , and 2theta is the scattering angle.

The radially averaged data exhibit a distinct flat region beyond Q = 0.35 Å-1 due to the incoherent scattering from hydrogen in the samples. This background scattering was subtracted from the data taken at both instrument configurations, normalizing for the difference in the protein concentrations. The background-subtracted data for both configurations were simultaneously corrected (desmeared) for instrument resolution effects, and Fourier transformed to obtain the distance distribution function, P(r), using two different methods: the Svergun method (16), as implemented in the program GNOM (17), and the Glatter method (18), as implemented in the program GLATTER, with resolution effects incorporated by Skov Pederson et al. (19). Both methods assume the scattering results from a dilute, monodispersed solution of globular particles. In addition, it is assumed that the particles have a sharp boundary such that there is a maximum diameter, Dmax, beyond which there is no significant scattering mass. The desmeared scattered intensity, I(Q), is related to P(r) by the equation,
I(Q)=4&pgr;V<SUB>0</SUB><LIM><OP>∫</OP><LL>0</LL><UL>D<SUB><UP>max</UP></SUB></UL></LIM>P(r)<UP>sin</UP>(Qr)/Qr dr (Eq. 2)
where the two conditions, P(0) = 0 and P(2r >=  Dmax) = 0, are satisfied. 4pi P(r) is defined as the number of distances, r, between scattering centers within the particle. The I(Q) versus Q data taken at both instrument configurations are combined in the desmearing process, and I(Q) versus Q data were obtained over a usable range 0.012 <=  Q <=  0.3 Å-1. The results from the two desmearing methods were averaged to produce the final desmeared I(Q) versus Q data and P(r) versus r data.

In the small angle limit, the scattered intensity can be approximated by the Guinier equation (13),
I(Q)=I(0)<UP>exp</UP><FENCE><UP>−</UP>Q<SUP>2</SUP>R<SUB><UP>g</UP></SUB><SUP>2</SUP>/3</FENCE> (Eq. 3)
where I(0) is the scattered intensity at Q = 0. The radius of gyration of the particle, Rg, is defined as the mean square distance from the center of scattering mass. A plot of ln(I) versus Q2 should contain a linear region with slope Rg2/3 and intercept ln(I(0)). Thus, Rg can be obtained directly from the SANS measurements. The range of validity for the Guinier approximation depends upon the shape of the particle. However, Guinier fits are generally made in a Q range centered about the relation, QRg ~ 1.0.

Details of the data desmearing procedure are illustrated in Fig. 1, which shows the smeared I(Q) versus Q data for the unligated CRP sample measured in D2O solvent. The dotted line is the best fit to the data using the Glatter (18, 19) method, in which the data are represented by a series of B-splines. The data were fitted several times using different estimates for Dmax. The fit that resulted in the smoothest P(r) function for which the Dmax value was clearly not too large, and for which the calculated I(Q) still fitted well to the experimental data, was chosen as the best solution. Dmax values of 65 ± 5 Å in Equation 2 proved to fit the smeared data best in all cases. The "x" points show the desmeared data resulting from the dotted line fit. Errors on the desmeared data were determined by averaging the best-fit results from both the Svergun (16, 17) and Glatter (18, 19) methods.


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Fig. 1.   SANS intensity versus Q data from 0.036 and 0.096 mM unliganded CRP in D2O solvent, uncorrected for instrument resolution effects (open circle ), along with the best fit obtained using the Glatter (18, 19) desmearing method (· · · ·), and the resultant desmeared data (×).

The I(Q) versus Q data obtained from SANS measurements can be compared with that calculated from the energy-minimized x-ray crystallographic structure using a Monte Carlo technique (20). Since hydrogen atoms are not included in the x-ray structure, each amino acid is better represented by a homogeneous sphere positioned according to its alpha -carbon coordinates. The size and neutron scattering strength of each sphere are calculated from the known volume and chemical composition of the amino acid, respectively, thus taking into account the contribution of hydrogen. Once a sphere has been drawn around each amino acid, a box is drawn around the entire molecule, with the option to include a region of bound water up to 5 Å thick. The bulk solvent can be any H2O/D2O mixture, and the protein can be hydrogenated or deuterated. Points are then generated at random within the box. The distance distribution function, P(r), for the total volume is calculated directly in real space by making a histogram representing the distances between all possible pairs of points, weighted according to the product of the neutron scattering strengths at each point. Rg is calculated directly from the second moment of P(r) and I(Q) is found using Equation 2.

Energy Minimization Computations-- The x-ray crystallographic structure of the cAMP-ligated CRP dimer shows that it can exist in an open and closed form in the crystal phase. The CRP dimers were generated from the Brookhaven Protein Data Bank file 3GAP.pdb exclusively in either the closed or open form by superimposing a copy of the monomer in the closed from on the monomer in the open form and vice versa (20). The energetics of the structures were then minimized using the steepest descent method for 1000 iterations, after which the conjugate gradient method was used until convergence was achieved (maximum derivative less than 0.04 kJ mol-1). Computational results were obtained using software programs from Biosym/MSI of San Diego. Energy minimizations, using the AMBER forcefield, were done with the Discover program, and structure analysis and comparison were performed with the DeCipher program. Energy-minimized structures for the unliganded CRP were generated in the same manner, using the cAMP-ligated x-ray structure, with the cAMP removed, as the starting structure.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The desmeared I(Q) versus Q data for the CRP and the cAMP-ligated CRP solutions are plotted in Fig. 2. It should be emphasized that at the levels of 0.036 mM CRP and 0.270 mM cAMP concentrations, the CRP is only about 90% ligated, so that the scattering would contain some contribution from unligated CRP. At higher cAMP concentrations of approximately 17.1 mM, where the CRP would be fully ligated (21), aggregation was observed in the CRP solutions as indicated by a sharp upward inflection of the neutron scattering intensity near Q = 0 and, thus, these data were not analyzed. The scattering curve for CRP in the presence of cAMP deviates significantly as shown in Fig. 2 from that of the unligated CRP above Q = 0.15 Å-1. This deviation results from a conformational change in CRP upon ligation with cAMP and is in contrast to the coincidence of the desmeared SANS data from solutions of CRP* in the absence and presence of 17.1 mM cAMP shown in Fig. 3. (Aggregation at this cAMP concentration was not observed for the CRP* solutions.) The nearly coincidental curves in Fig. 3 show that CRP*, contrary to CRP, undergoes very little conformational change in the presence of cAMP, as implied by the in vivo activation of transcription by CRP* in the absence of cAMP.


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Fig. 2.   Desmeared SANS intensity versus Q data from unliganded CRP in D2O solvent (bullet ) and from CRP + 0.270 mM cAMP in D2O solvent (), where the CRP is about 90% in the cAMP-ligated form.


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Fig. 3.   Desmeared SANS intensity versus Q from 0.049 and 0.138 mM unligated CRP* (bullet ) and CRP* + 17.1 mM cAMP () in D2O solvent.

The radii of gyration calculated from the second moment of the distance distribution function, P(r), using all of the desmeared data in Figs. 2 and 3 are presented in Table I. For comparison, Table I also lists the radii of gyration obtained by averaging the results from several Guinier fits over the Q range limited to 0.02 <=  Q <=  0.08 Å-1 for unligated and ligated CRP and CRP*. The radii of gyration calculated by the two methods are essentially the same for the unligated and cAMP-ligated CRP* solutions (approximately 22.3 ± 0.3 Å), indicating again virtually no difference between the size of CRP* and cAMP-ligated CRP*. This is in contrast with the results from the CRP solutions, using the two methods, which exhibit a consistent shrinkage of about 1.0 Å upon cAMP ligation. The slightly larger Rg values determined for CRP from the Guinier fits compared with those determine from the 2nd moment of P(r) may be due to the effect of some aggregation in the sample on the Guinier-derived Rg. The Rg values calculated from P(r) are not significantly affected by small deviations in the scattering curve due to aggregation effects at small Q, where the Guinier-derived Rg values were determined.

                              
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Table I
Comparison of the Radii of Gyration (Rg) for CRP and CRP

The desmeared data in Fig. 2 are compared in Fig. 4 to model scattering curves calculated from their corresponding energy-minimized x-ray structures, using 2000 randomly placed points in the volume occupied by the molecule. Three iterations were performed for each of four structures, i.e. unligated CRP dimer in the exclusively open and closed forms, and cAMP-ligated CRP dimer in the exclusively open and closed forms. The final model curves represent the average of the three iterations. The unligated CRP scattered intensity, along with the model intensities calculated from the energy-minimized x-ray structures of CRP with cAMP removed, are plotted in Fig. 4A as a function of the dimensionless parameter, Q·Rg (QRg). Plotting the curves in this manner maximizes the differences in structure due to changes in overall shape, while minimizing changes in Rg. In Fig. 4A, the experimental desmeared SANS data fit very closely the model curve for unligated CRP exclusively in the open form. The close agreement between the desmeared SANS data and the model curve of CRP in the open form indicates that the open form of CRP is the preferred conformation of unligated CRP in solution. This is further substantiated by the radii of gyration obtained from the second moment of P(r) for the energy-minimized x-ray structures listed in Table I. The Rg value from the desmeared unligated CRP data is 22.0 ± 0.2 Å, which compares more favorably with 20.5 ± 0.4 Å for the minimized, exclusively open form of CRP than with 18.7 ± 0.3 Å for the minimized, exclusively closed form of CRP.


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Fig. 4.   Desmeared SANS intensity versus QRg data from unligated CRP (A) and cAMP-ligated CRP (B) in D2O solvent (bullet ), along with model SANS curves calculated from energy-minimized x-ray structures of unligated CRP (A) and cAMP-ligated CRP (B) in the closed form (open circle ) and in the open form ().

The desmeared scattered intensity for cAMP-ligated CRP is plotted in Fig. 4B as a function of QRg, along with the model intensities obtained from the energy-minimized x-ray structures of cAMP-ligated CRP. The model curves for the minimized exclusively closed and open forms of the cAMP-ligated CRP are less separated than the corresponding model curves for unligated CRP in Fig. 4A and have Rg values of approximately 19.2 ± 0.3 Å in each case, compared with Rg = 21.6 ± 0.2 Å for the desmeared data in Table I. However, the desmeared data appear to follow more closely the model curve calculated from the minimized structure of cAMP-ligated CRP dimer exclusively in the closed form.

The SANS results show that the conformational change that occurs in CRP upon ligation with cAMP can well be described as a change from the open to the more compact closed form of CRP. This is further evident in the distance distribution functions, P(r), plotted for unligated and cAMP-ligated CRP in Fig. 5A. In the presence of cAMP, the peak shifts slightly toward smaller r values, indicating that cAMP-ligated CRP is more compact than nonligated CRP. Fig. 5B shows the P(r) functions obtained from the energy-minimized x-ray structures that fit most closely to the data. The distributions look remarkably similar to those in Fig. 5A, with the peak in the distribution for the cAMP-ligated CRP in the closed form occurring at a lower value than that for the nonligated CRP in the open form.


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Fig. 5.   Distance distribution functions obtained from the desmeared data for unligated CRP (bullet ) and cAMP-ligated CRP () in D2O solvent (A) and from energy-minimized x-ray structures (B) of unligated CRP in the open form (bullet ) and cAMP-ligated CRP in the closed form () (A).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The SANS results show that ligation of CRP by cAMP apparently induces a conformational change in CRP from the exclusively open form in solution to the more contracted closed form, whereas CRP* remains essentially unchanged in the presence of cAMP in solution. It should be emphasized that the energy-minimized open form of unligated CRP is based on energy minimization of the x-ray crystal structure of cAMP-ligated CRP, exclusively in the open form, with cAMP removed, because the x-ray crystal structure of unligated CRP is unavailable. The good agreement between this conformation of unligated CRP and the experimental SANS curve does imply that this energy-minimized open form may indeed be the stable conformation of unligated CRP in solution. These results agree with NMR measurements, which show a tightening up of CRP into a more rigid protein upon cAMP binding (2). The magnitude of the change of approximately 1 Å in the radius of gyration also agrees with the 0.7 ± 0.1 Å shrinkage observed in the Stokes radius of CRP upon ligation with cAMP (22). Although the change in CRP is in agreement with earlier small angle x-ray scattering measurements, which also indicate a contraction in the shape of CRP upon binding of cAMP, the x-ray measurements exhibited a larger change in Rg from 29 Å for unligated CRP to 25 Å for the cAMP-ligated CRP complex (5). Calculations on the energy-minimized unligated and ligated CRP structures show that Rg would contract only from 20.5 Å for the open form of CRP to 19.2 Å for the closed form of the complex. The x-ray scattering results show not only a large change in Rg, but also an absolute Rg value 6-9 Å larger than could be calculated from the x-ray crystallographic structures. The presence of aggregates in the small angle x-ray scattering samples could easily account for the higher Rg values and larger relative change in Rg in the presence of cAMP.

The change to the closed form for the cAMP-ligated CRP complexes in solution also agrees with molecular dynamics simulations on the cAMP-ligated CRP complex, which predict that the closed form is the more energetically stable form of this complex in solution (9). In addition, the x-ray crystallographic structure of the cAMP-ligated CRP complexed with DNA shows that the cAMP-ligated CRP exists exclusively in the closed form for this ternary complex (8). Lack of growth for CRP crystals in the absence of cAMP may arise from the CRP in solution being exclusively in the open form.

The energy-minimized structures for unligated CRP in the open form and cAMP-ligated CRP in the closed form are shown in Fig. 6, which illustrates the small, but significant, structural differences between the two forms. A model proposed by Garges and Adhya (23) for the conformational change of CRP upon cAMP binding involves four structural changes in CRP: 1) a change in the orientation of the D alpha -helix (Fig. 6), 2) an interaction between the cAMP and DNA binding domains, 3) re-alignment of the CRP monomers, and 4) more importantly, the "pushing away" of the DNA-binding F alpha -helices (Fig. 6) from the protein. These changes also allow the two F alpha -helices to bind into two adjacent grooves of DNA. The changes described by the model with respect to the interaction between the cAMP and DNA binding domains and a change in the distance between the DNA-binding F alpha -helices are also shown in Fig. 6 in the transition from the unligated open form of CRP (Fig. 6A) to the ligated closed form of CRP (Fig. 6B). The distance between the F alpha -helices in the closed form is more conducive to DNA binding as evident by the cAMP-CRP-DNA crystal structure, which is exclusively in the closed form. Thus, the changes described in this model support the SANS results, which show a conformational change from unligated CRP in exclusively the open form to the closed form upon cAMP ligation in solution.


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Fig. 6.   Energy-minimized structure for nonligated CRP in the open form (A) and for cAMP-ligated CRP in the closed form (B).

The lack of significant changes in the CRP* scattering curves upon complexation with cAMP implies that CRP* undergoes little, or possibly no, structural change in the presence of cAMP. This is in agreement with the observation that transcription is activated in the absence of cAMP in cells which contain CRP* (11), implying that unligated CRP* is already in the correct conformation for binding of the DNA. To substantiate that this conformation is the closed form, fits of the energy-minimized structure of CRP* to the SANS data must await the determination of a good starting structure for CRP* from x-ray crystallography.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB-9722884 (to F. P. S.) and National Science Foundation Grant DMR-9423101 (to the National Institute of Standards and Technology Center for High Resolution Neutron Scattering).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 301-738-6272; Fax: 301-738-6255.

The abbreviations used are: CRP, cAMP receptor protein; CRP*, T127L,S128A mutant of CRP; SANS, small angle neutron scattering measurements.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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