1 Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA and 2 Laboratoire de Biochimie Théorique, CNRS UPR 9080, Institut de Biologie Physico-Chimique,13 rue Pierre et Marie Curie, 75005 Paris, France
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Abstract |
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Keywords: DNA bending/free energy/molecular modeling/protein-DNA complex/p53
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Introduction |
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Human p53 consists of 399 amino acids and has been divided structurally and functionally into four domains: the N- and C-terminal domains, the p53 DNA binding domain (p53DBD) and the tetramerization domain. The structures of the two last domains have been resolved, respectively, by X-ray (Cho et al., 1994) and NMR methods (Clore et al., 1994
, 1995
; Lee et al., 1994
; Jeffrey et al., 1995
; Kuszewski et al., 1999
). The structure of a short sequence of 11 amino acids residues from the N-terminal domain has also been released (Kussie et al., 1996
). However, this structural information does not determine the overall arrangement of the domains relative to each other in the functional multimers. The N-terminal domain, comprising the first 44 amino acids, is a transcriptional activation domain that interacts with the basal transcription machinery in positively regulating gene expression (Unger et al., 1992
; Chang et al., 1995
). The p53DBD (residues 102292) forms a tetrameric complex with DNA, but only the structure of a monomeric p53DBDDNA complex has been determined (Cho et al., 1994
). The core domain structure consists of a ß-sandwich that serves as a scaffold for two large loops held together in part by a tetrahedrally coordinated zinc and a loopsheethelix motif. Residues of the loopsheethelix motif interact directly in the major groove of DNA and an arginine from one of the two loops penetrates the minor groove (Cho et al., 1994
); 90% of the missense mutations in p53 residues are located in the p53DBD. The X-ray structure has been very useful in providing information about the role of these mutations and led to their division into two classes (Cho et al., 1994
). Mutations of the first class result in defective contacts with the DNA and the loss of the ability of p53 to act as a transcription factor. A second class of p53 mutations disrupts the secondary structure, altering the conformation of the protein.
The p53DBD is linked to the tetramerization domain (residues 342355) by a flexible linker of 37 amino acids (287323). The tetramerization domain is a dimer of dimers (Clore et al., 1994, 1995
; Lee et al., 1994
; Jeffrey et al., 1995
; Kuszewski et al., 1999
). Each monomer contributes to the dimer complex by one ß-strand and one helix, across an anti-parallel ß-sheet and helixhelix interface. The two dimers interact via a second parallel helixhelix interface, forming a four-helix bundle. The C-terminal domain of p53 is composed of nine basic amino acid residues that bind to DNA and RNA with sequence and structural preferences. The C-terminal domain either sterically or allosterically regulates the ability of p53 to bind to specific DNA sequences at its central core. The wild-type p53 protein seems to have two conformations, one latent and one active that can be detected in vivo (Hupp and Lane, 1994
). The latent form of the protein appears to be maintained by the C-terminal basic region. The alteration of this domain can activate p53 for DNA binding (Hupp and Lane, 1992; Halazonetis et al., 1993
).
Wild-type p53 binds over 100 different naturally occurring response elements but only approximately 50 show functionality (Tokino et al., 1994). Response elements differ in the details of their base sequence, but all consist of two tandem decameric elements, each a pentameric inverted repeat (
, where
represents a pentamer). The sequence of most decamers closely follows the consensus pattern: PurPurPur(C(a/t)|(a/t)G)PyrPyrPyr [where Pur represents a purine and Pyr a pyrimidine (el-Deiry et al., 1992
) and the central bar indicates the center of pseudosymmetry]. The decameric elements may be separated by as much as 21 base pairs without complete loss of p53 binding affinities, but functional sites have no spacers or at most very short ones (Tokino et al., 1994
). X-ray crystallography has revealed that one core domain monomer of p53 binds to one pentamer, suggesting that the p53DBD monomers are arranged in a head-to-head orientation (Cho et al., 1994
).
No structure is available for the tetrameric complex of the p53DBD units with the DNA. However, a first model has been proposed by Cho et al. (1994), who constructed a model of the tetramerDNA complex using straight B-DNA and details from the monomeric p53DBDDNA complex. The model suggested that (i) tetrameric binding is possible, as there were no steric clashes observed between the monomers in this arrangement, and (ii) that additional proteinprotein contacts can be made between adjacent monomers through the H1 helices.
Complementary structural information about p53 binding to DNA was obtained from circular permutation experiments (Nagaich et al., 1997). The tetrameric p53DBD was found to bend the DNA in a manner dependent on the sequence of the four base pairs localized at the junction of two head-to-head pentamers (C(a/t)|(a/t)G). These sequences are most commonly CATG, but CAAG and CTTG also occur. All are known from the literature to exhibit a large flexibility for bending or kinking toward the major groove (Satchwell et al., 1986
; Bolshoy et al., 1991
; Schultz et al., 1991
; Zhurkin et al., 1991
; Mauffret et al., 1992
; Mujeeb et al., 1993
; Gorin et al., 1995
). The authors observed that the bending is higher, ~50°, if the sequence is CATG and is between 37° and 25° for the CTTG sequence. Moreover, electrophoretic mobility shift assays show a direct correlation between bending and affinity. The stability of the complex becomes greater the more the DNA bends (Nagaich et al., 1997
). These results suggest that both the structure and the stability of the p53DNA complex may vary with different response elements. An A-tract phasing analysis found significant differences between the bending and twisting of DNA (AGGCATGCCT) by p53DBD and by the full-length wild-type p53 (Nagaich et al., 1999
). These experiments allowed the determination of the magnitude and directionality of the bending. p53DBD bends the DNA by 3236°, whereas the wild-type p53 bends it by 5157°; both bend the DNA toward the major groove at the center of the binding site.
Based on the assumption that the DNA bending is localized at the junction between the pentamers, a second tetrameric model of p53DBDDNA complex was proposed more recently (Durell et al., 1998). A series of structures were constructed for a range of DNA conformations resulting from correlated changes in bends and twist at the junction between head-to-head pentamers. A narrowing of the minor groove was found to occur as the DNA is bent toward the minor groove, inducing steric clashes between the phosphodiester backbones of the DNA. On the other hand, the models suggested that DNA bending toward the major groove, as observed experimentally, forms a reasonable interface between the two adjacent p53DBD monomers. The authors assumed that the p53DBDDNA complexes are stabilized by interactions between monomers, explaining the cooperativity of multimeric p53 binding to DNA observed experimentally (McLure and Lee, 1998
).
To explore some of the energetic aspects of this complex and its structural characteristics, we developed the modeling procedure described here for the dimeric p53DBDDNA complex in which a flexible construct is used to explore the conformations of the DNA at the pentameric junction. The X-ray structure of the monomeric p53DBDDNA complex serves to define a `molecular mold' and the conformation of DNA is obliged to fit this mold. The conformational space available for the formation of p53DBDDNA dimer was explored within the structural constraints of the monomeric complex. For each conformation of the complex explored, the electrostatic free energy of formation and the van der Waals interaction energy were calculated with DELPHI and CHARMM and compared with the proteindimer without the DNA. We evaluated the suggestion made earlier (Durell et al., 1998) that the interface between the monomers is a driving force in the formation of the complex and can explain the cooperativity of multimeric p53DBD binding to DNA (McLure and Lee, 1998
). We show that the most stable complex appears to be that with a DNA bend of 11°, in reasonable agreement with the experimental results. However, in contrast to inferences from the earlier model, the free energy calculations show that the DNA strongly assists the formation of the complex as the dimer is unstable by itself in solution.
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Methods |
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The approach involves three main steps: first a template or `molecular mold' is created defining the local DNA conformation involved in binding a protein. In the second step, the DNA is deformed to comply with the constraints of the local conformations defined by one or more molds and an energy minimization is carried out within JUMNA (JUnction Minimization of Nucleic Acids) (Lavery et al., 1995). In step three, the protein or protein components are repositioned on the deformed DNA according to structural constraints from experimental data.
The program JUMNA differs from most molecular modeling approaches in that it represents DNA using a combination of internal and helical coordinates, rather than Cartesian atomic coordinates. All bond lengths are taken to be fixed and valence angle changes are limited to the phosphodiester backbones and sugar rings. Helicoidal coordinates are introduced by breaking the nucleic acid into a series of 3'-monophosphate nucleotides. Each nucleotide is positioned in space with respect to a helical axis system, using three translational (Xdisp, Ydisp, Rise) and three rotational (Inclination, Tip, Twist) variables. The internal movements of the nucleotides are represented by sugar puckering (four variables), the glycosidic bond, the two bond torsions (, C3'O3' and z, O3'P) and two valence angles (C3'O3'P; O3'PO5') within the phosphodiester backbone. Internucleotide links (O5'C5') are maintained using quadratic distance constraints. The bond rotations and valence angles involving these linkages are dependent variables. JUMNA uses the FLEX force field, developed specially for nucleic acids (Lavery et al., 1995
). This allows the calculation of conformational enthalpies, which include LennardJones and electrostatic energies of interaction between non-bonded atoms (including an angle-dependent hydrogen bonding term) as well as contributions from valence angle and bond torsion terms. Solvent damping of electrostatic interactions is treated using a sigmoidal distance-dependent dielectric function (Hingerty et al., 1985
) and counterion binding is mimicked by reducing the net phosphate charge (0.25e being added to the point charge on each anionic oxygen). Although this is a simple model, which ignores detailed solvent, salt effects and entropic features, it has been found to yield structurally valid double helical structures and to predict conformational transitions, in good agreement with experimental data (Cluzel et al., 1996
; Sanghani et al., 1996
). Finally, this representation of DNA permits any structural feature of the model to be controlled very simply, by driving any kind of deformation such as stretching (Cluzel et al., 1996
; Lebrun and Lavery, 1996
), unwinding or bending (Lebrun et al., 1997
), using specific restraints.
The first step of the modeling algorithm is carried out using the program CONTACT which constructs a `molecular mold' defining the protein-binding interface on a DNA molecule. CONTACT reads the coordinates of a proteinDNA complex and looks for the DNA atoms involved in the protein-binding interface using a cutoff distance between DNA and protein atom pairs defined by the user (4 Å in the present case). Flexibility can be introduced by defining the radius of a sphere around the experimentally determined position of each DNA atom, inside which the atom will be allowed to move during the subsequent energy minimizations (generally 0.2 Å). A similar freedom can be introduced by using a small force constant to constrain the atomic positions (25 kcal/mol.Å2). CONTACT then generates a set of restraints defining the position of the DNA interface atoms in space subject to the chosen precision. Optionally these N restraints may be transformed into a set of N(N 1)/2 interatomic distance restraints which define the same protein-binding interface, but whose position in space is no longer fixed.
The second step involves the program JUMNA that can be used to deform a fragment of DNA so that its conformation respects the restraints defined by CONTACT. In the case where atomic position restraints are used, we start by superposing the set of atomic positions defined by CONTACT with the initial DNA conformation built by JUMNA so as to obtain a minimal root mean square difference. At the end of the JUMNA minimization, the inverse transformation can be used to place the deformed model DNA in the axis system of the proteinDNA complex. If alternatively interatomic distances are used (allowing the DNA to move in space), no such transformation is necessary, but a utility program, CLIP, is available to position the protein from the initial complex on the binding interface of the DNA molecule created by JUMNA. Whichever approach is chosen, the set of restraints defining the `molecular mold' can also be translated into equivalent restraints acting on a dyad-related binding site.
All the DNA structures were analyzed with CURVES (Lavery and Sklenar, 1988, 1989
). The results presented here include local variables, which describe the relative position of successive base pair steps and global variables related to a curvilinear helical axis. Molecular structures were displayed with InsightII 98.0 (Biosym/MSI).
Dimerization interface and free energy of formation
The modeled structures of the p53DNA complex were energy minimized with the CHARMM molecular mechanics program (Brooks et al., 1983; MacKerell et al., 1995
), keeping the DNA conformation fixed. A recent study of proteinprotein association, comparing experimental structures of the monomers and protein dimers, shows substantial changes in the side chain as well in the main chain conformations during the association process (Betts and Je, 1999
). Consequently, we used a two-step minimization procedure, first restraining the protein backbone with a small force to the X-ray structure and then releasing the restraints. During the second step the minimization was carried out to a gradient value of 0.05. The DNA structure was kept fixed throughout to respect the `molecular molds' defined from the interaction with the X-ray structure. However, minimization with 100 steps of steepest descent (SD) and 100 steps of ABNR was performed on the DNA for each minimized complex to relieve any stress related to the change of the force field (FLEX to CHARMM24). The monomers from the X-ray structure of the p53DBDDNA complex were used to model the first complex that was minimized as described before and these minimized monomers were then used to construct the subsequent complex in which the DNA had a range of bending angles. The same minimization procedure was performed for each complex. Given the size of the systems treated and the large number of restraints imposed, it is generally difficult to minimize to a better gradient tolerance than 0.05. However, a number of tests performed with a tolerance of 0.03 suggest that the energies presented here are defined to a precision of roughly 10 kcal/mol.
For each complex the total free energy of complex formation in water was calculated as the sum of the van der Waals energy of interaction and the total free electrostatic energy of binding in solution. The total free electrostatic energy is defined as the sum of the direct Coulomb interactions of each single charge with all other charges in the system and the interaction of each charge with its self-reaction field and the reaction field of the other charges. The total free electrostatic energy of binding is defined here as the difference between the total electrostatic energy of the complex and that of the separate components. We used the minimized X-ray monomer and a minimized straight B-DNA as the reference state for the separated components. The monomer from the X-ray coordinates was minimized in CHARMM using the same procedure as for the complex. The straight B-DNA was constructed and minimized in JUMNA, then minimized in CHARMM (100 SD + 100 ABNR steps) to relieve any steric clashes related to the change of the force field.
We used finite difference solutions to the linearized PoissonBoltzmann equations in DELPHI to calculate the total electrostatic free energy in solution (Gilson et al., 1985; Gilson and Honig, 1988
). For these calculations, the protein was treated as a low-dielectric medium (
= 4) delimited by the molecular surface calculated from the rolling sphere procedure with a probe of 1.4 Å radius. It was surrounded by a region with a high dielectric constant (
= 80). The ionic strength was set to the physiological value of 0.145 M and the Stern layer radius to 2 Å. Atomic charges and radii were taken from the CHARMM24 force field. The reaction field calculation was performed with the focusing option (Gilson and Honig, 1988
) that utilized boundary potentials from a previous run, with a course grid (the starting percentage fill was 20%, focused to 90%). Using a cubic grid of 201 grid points per side, we obtained a resolution of at least 2 grid points/Å. The initial boundary conditions imposed on the edge of the lattice were approximated from the DebyeHückel potential. It should lastly be added that free energies calculated in this way do not include the terms related to the formation of hydrophobic cavities or entropy, which can be expected to be favorable and unfavorable, respectively, to complex formation.
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Results |
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The proteinDNA contacts in the p53DBDDNA complex were established with the program CONTACT as described in Methods. Inspection of the monomeric p53DBDDNA crystal structure shows that the monomer interacts not only with the five base pairs AGGCT, but also with some of the quasi-symmetric ones (Cho et al., 1994). In particular, at the pentameric junction, Arg248 interacts in the minor groove with one DNA strand of each pentamer (see Figure 1
, adapted from the paper by Cho et al., 1994). Consequently, upon dimerization, the introduction of a second Arg248 in the minor groove will affect the conformation of both pentamers at the junction. This observation suggests that the interactions found for the co-crystal structure at the four central base pairs CTTG will change, at least partially, upon dimerization. Nevertheless all atoms considered to interact with the monomer in the crystal structure were kept in the restraints defined by CONTACT. These restraints were used to define a fixed `mold' for the first pentamer and were translated with the dyadic symmetry of the p53 consensus sequence, to create a second free `mold' (that is, free to move in space).
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To explore the formation of the dimerization interface as a function of the DNA structure, the bending of the DNA was driven with JUMNA by introducing an axis break between the monomer binding sites. The six variables relating the two segments are the Ax, Ay, Rise, Ainc, Atip and Twist (Lavery et al., 1995). The Atip parameter allows control of the bending toward the grooves. Bending was performed through a steady change of this variable (5° per step) followed by an energy minimization at each step. The WatsonCrick hydrogen bonds of the central base pairs CATG were constrained throughout in order to prevent their opening. After each step, the p53DBD monomers were repositioned on their sites within the deformed DNA (see Methods).
Monomer interfaces as a function of DNA bending
The DNA is bent at the pentameric junction as described in Methods and minimized at each step. The interface surface is measured as the difference between the surface of the monomers and the surface of the dimer, as a function of the DNA bending. Figure 2 shows that a DNA bending angle of 21° places the monomers too far apart to interact effectively. For this DNA geometry the calculated interface between the two monomers is only ~400 Å2. This value is much less than the lower limit value of ~750 Å2 observed in X-ray structures for protein interfaces (Jones and Thornton, 1996
). As DNA bending toward the major groove decreases, the monomers come closer to each other and the interface reaches a plateau of ~800 Å2, around a DNA bending angle of 14°. Below 7° DNA bending the monomers are too close, creating bad steric clashes. Moreover, a negative bend toward the minor groove causes a groove narrowing, leading to repulsive contacts between the DNA phosphates as already observed in a previous modeling study (Durell et al., 1998
). Consequently we limited further analysis to the DNA structures that allow acceptable interactions between the monomers, that is, with DNA bending toward the major groove in the range 621°.
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The van der Waals energy of interaction and the free electrostatic energy of binding were examined separately in order to evaluate the role of the different energy components in the formation of the complex. These components were calculated for the dimeric p53DBDDNA complex as well as the p53DBD dimer to gain insight into the driving forces of complex formation.
Figure 3a and b display, respectively, the electrostatic free energy of binding of the uncomplexed free dimer calculated with DELPHI and the van der Waals energy of interaction obtained with CHARMM as a function of DNA bending angle. The dependence of their sum on the DNA bending angle is shown in Figure 3c
.
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The right panels in Figure 3 (d, e and f) display, respectively, the total electrostatic free energy of binding for the dimeric p53DBDDNA complex calculated with DELPHI, the van der Waals energy of interaction obtained with CHARMM and the total free energy of binding as a function of the DNA bending.
As for the free dimer, the electrostatic free energy of binding of the complex increases linearly as the DNA is straightened. However, in this case, the electrostatic energy remains generally favorable to complex formation. This is due to the decrease in the reaction field generated by the complex compared with the components. Because the large positive charged interface is neutralized by the association with the negatively charged phosphates of the DNA, the reaction field generated by the dimer in the complex is smaller than that induced by the separated components (Figure 5).
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We have also looked at the decomposition of the protein binding energy along the target DNA sequence, by comparing the binding of each monomer with the corresponding target DNA sequence and the dimeric complex. This was done in order to determine if there is a cooperative effect of monomer binding in terms of DNA deformation energy. The energy of a given DNA conformation can be decomposed by nucleotide pairs, by calculating the internal energy of the pair in question plus half the interaction energy of this pair with the rest of the structure (the sum of these terms being equal to the total energy of the oligomer). It is similarly possible to calculate the decomposition of deformation energy simply as the difference of the nucleotide pair contributions between the bound and unbound DNA conformations.
The results for the initial p53 dimer complex we have created are shown in Figure 6. The energy curve along the target sequence shows that the only significant deformations occur within the adjacent pentamer sites and that the largest values involve the central GCATGC segment. It can be noted that within the GCA fragment of each pentamer the deformation energy is higher at the G and A nucleotide pairs than at the intervening C, which may be related to the absence of proteinnucleic acid backbone contacts at this position. If we again subdivide the deformation energy into its two-monomer contributions (Figure 6
) it is confirmed that each monomer only significantly deforms its own pentamer-binding site. These data also suggest that dimer binding is not cooperative from the point of view of the DNA deformation energy, since the total deformation energy curve of the dimer is almost identical with the curve obtained by adding the individual monomer deformation energies.
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Figure 7 illustrates the complexes for DNA structures with 21°, 11° and 7° bending (the protein is represented by a thick blue ribbon and the DNA backbone by a yellow ribbon). Arg248 is shown penetrating the DNA minor groove in all the frames. The top picture shows the complex for a DNA bending of 11° from a side view. As evidenced in the figures, the monomers interact through the H1 helices. In the structure corresponding to a bend of 11° toward the major groove (Figure 7b and e
), the two H1 helices form an anti-parallel complex stabilized by two salt bridges between the two Arg181 and the two Glu180 residues. The rings of Pro177 and His178 are close enough to have an edge-to-face contact. In addition, His178 is also close enough to the carbonyl group of the Met243 to have a close contact.
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As the bending of the DNA increases (Figure 7a and d), the H1 helices are moved apart. For a DNA bending of 21°, the Glu180 and the Arg181 are now more than 5 Å apart. However, His178 and Met243, as well as the Pro177, are still close enough to interact.
The initial conformation of the DNA deformed to respect the p53DBD binding interfaces was bent by 21° toward the major groove owing to a strong positive roll of 18°, localized at the junction between the pentamers (ApT step) (Figure 8a). The others steps had only small roll values, ranging from 0° to 3°. As the DNA bending decreases, the roll at the ApT step changes by only 4°. The variation in roll occurs mainly at the CpA and the symmetric TpG steps, which appear to be the most flexible. They both vary from 0° to 10° compensating the ~15° roll of the ApT step as the DNA straightens. The p53DBD binding interface restraints locally produce a narrowing of the minor groove as CpA and TpG tend to negative roll values. These compressions of the minor groove favor the interactions of the two Arg248 residues with the two TpG steps and could explain the protection against the cleavage observed experimentally (Nagaich et al., 1997
). In addition, the roll values observed here are in a range corresponding to the CA- family, a subdivision of the two CpA dinucleotide conformational families described from X-ray B-DNA structures (Gorin et al., 1995
). In contrast, the strong positive roll at the ApT step enlarges the minor groove and maintains an overall bending of the DNA toward the major groove. These results are also consistent with the A-tract phasing experiments showing a bending toward the major groove presumably localized at the CATG junctions (Nagaich et al., 1999
).
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Discussion |
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The modeling procedure presented here allows the formation of a DNA structure with an r.m.s.d. for all atoms of 0.5 Å compared with the X-ray pentamer by using constraints on the DNA atoms contacted by the protein. These DNA structures can be recombined with the protein monomer, preserving the interactions between the DNA atoms and the amino acids of the p53DBD monomer observed in the X-ray structure of the complex. Consequently, we were able to consider the formation of the dimer as a function of DNA bending and also to examine the changes in the local DNA structure of the CATG sequence upon bending. In an earlier study, Durell et al. (1998) proposed a new model of the p53DBDDNA complex based on a DNA structure having an average value of 33.6° and 3.1 Å for the twist and rise parameters, respectively, and setting the other helicoidal parameters to zero (Durell et al., 1998). By superimposing each DNA pentamer with the monomeric p53DBDDNA X-ray complex, the p53DBDDNA complex was formed with the average DNA, upon deletion of the original DNA from the X-ray structure. In this way, an r.m.s.d. of 0.7 Å was obtained for the C1' atoms in compared with the experimental DNA structure.
The main features of the interactions of the monomeric protein with DNA (Figure 1) are (i) the GGC bases contacted by amino acid side chains in the major groove, (ii) the Arg248 inserted into the minor groove and (iii) several amino acids interacting with the DNA backbone. The DNA has a straight B-like conformation and the authors note a slight compression of the minor groove which allows the tight packing of Arg248 again the sugar and the phosphate groups inside the minor groove (Cho et al., 1994
). Hydroxyl radical cleavage experiments show that the minor groove is relatively narrow at the CATG pentamer junction and show a protection of the adjacent TG dimers, possibly as the result of the interaction of the two Arg248 with these bases (Nagaich et al., 1997
). In contrast, A-tract phasing experiments show a bending toward the major groove supposedly localized at the CATG junction (Nagaich et al., 1994). In this case, one is more likely to observe an opening of the minor groove instead of the compression described for the X-ray structure. The co-crystal structure reveals that the core domain of p53 binds principally to a single pentamer consensus sequence, but also to part of the adjacent pentamer at the pentamer junction. In modeling the dimer, we assumed that when the second monomer binds the DNA, it produces complementary interactions with the phosphate backbone at the pentamer junction that will change the local conformation. These dimerization-related interactions could reasonably be expected to affect the structure at the pentameric junction compared with that one observed in the crystal (Cho et al., 1994
).
The change in the structural parameters associated with the bending of DNA in the complex (see Results) is reflected in the behavior of the ApT and CpA/TpG steps of DNA (Figure 8a). The nature of the structural rearrangement becomes evident from the comparison to the free DNA target sequence (Figure 8b
). In this case, the majority of the bending comes from the CpA and TpG steps, as would be expected from earlier studies of sequence-dependent flexibility (Sarai et al., 1989
; Gorin et al., 1995
; El Hassan and Calladine 1996
; Olson et al., 1998
). While the central ApT step still increases its roll as the DNA bends, the magnitude of the roll lies >20° below the values induced in the protein complex.
The free energy of binding of the dimer indicates the formation of a stable dimer when the DNA is bent toward the major groove by about 11°. This result is consistent with the gel retardation experiments, in which the p53DBDDNA and the entire p53DNA complexes were phased with respect to the curvature of A-tracts: The four subunits of p53DBDB bend the DNA by 3236°, implying 1618° per half-site (Nagaich et al., 1999). The structures presented here are consistent with those proposed earlier (Durell et al., 1998
). Two structures were described corresponding to a DNA bending toward the major groove, one at a global minimum at 7.5° of DNA bending and another with an energy +30 kcal/mol higher, but at a DNA bending of 20° closer to the experimental value. In the case of the 7.5° complex proposed by Durell et al. (1998), the H1 helices form an anti-parallel complex allowing two salt bridges between the Glu180 and the Arg181 residues. Below the helices, the Pro177 and His178 form edge-to-face contacts. The second structure, proposed by these authors, corresponding to a higher positive DNA bending, shows a greater separation of the H1 helices, breaking the two Glu180Arg181 salt bridges. This is compensated by enhanced inter-subunit complex interaction comprising Pro177, His178 and Met243. The His178 residues adopted parallel stacked orientations. The first structure of 7.5° is very similar to that described in Figure 7c
for a DNA bending of 7°. The second structure of 20° bend is similar to those described in Figure 7b and a
, respectively, for a DNA bending of 11° and 21°. The differences in the values of bending obtained for similar dimer structures can be explained by the difference in the methods used to construct the proteinDNA monomers, as well as to bend the DNA. Note that constraints imposed by p53DBD binding lead to the DNA bending mainly via the ApT site at the pentamer binding site junction rather than through the adjacent and normally more flexible CpA/TpG sites.
The clearest inference from our energy calculations is that the DNA strongly supports complex formation, as the protein dimer is unstable by itself in solution. Not only do the DNA interactions with the protein side chains produce a favorable van der Waals energy, but also the DNA is found to neutralize the strong positive electrostatic potential generated by monomer association. This stabilizes what would normally be an unfavorable interaction in solution (see Figures 4 and 5). Decomposition of the protein binding energy along the target DNA sequence, however, shows that dimer binding is not cooperative in terms of DNA deformation energy (see Figure 6
).
The technique we have developed for constructing proteinDNA binding interfaces, as well as the energy decomposition approaches used in the analysis of the resulting model, are generalizable. Using cognate structural information combined with other types of experimental data about the constituent binary interactions, the techinque offers the exciting possibility of assembling multi-component proteinDNA complexes.
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References |
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Received August 8, 2000; revised December 11, 2000; accepted January 3, 2001.