Structures of Zinc Finger Domains from Transcription Factor Sp1
INSIGHTS INTO SEQUENCE-SPECIFIC PROTEIN-DNA RECOGNITION*

(Received for publication, August 13, 1996, and in revised form, November 15, 1996)

Vaibhav A. Narayan , Richard W. Kriwacki § and John P. Caradonna

From the Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The carboxyl terminus of transcription factor Sp1 contains three contiguous Cys2-His2 zinc finger domains with the consensus sequence Cys-X2-4-Cys-X12-His-X3-His. We have used standard homonuclear two-dimensional NMR techniques to solve the solution structures of synthetic peptides corresponding to the last two zinc finger domains (Sp1f2 and Sp1f3, respectively) of Sp1. Our studies indicate a classical Cys2-His2 type fold for both the domains differing from each other primarily in the conformation of Cys-X2-Cys (beta -type I turn) and Cys-X4-Cys (beta -type II turn) elements. There are, however, no significant differences in the metal binding properties between the Cys-X4-Cys (Sp1f2) and Cys-X2-Cys (Sp1f3) subclasses of zinc fingers.

The free solution structures of Sp1f2 and Sp1f3 are very similar to those of the analogous fingers of Zif268 bound to DNA. There is NMR spectral evidence suggesting that the Arg-Asp buttressing interaction observed in the Zif-268·DNA complex is also preserved in unbound Sp1f2 and Sp1f3. Modeling Sp1-DNA complex by overlaying the Sp1f2 and Sp1f3 structures on Zif268 fingers 1 and 2, respectively, predicts the role of key amino acid residues, the interference/protection data, and supports the model of Sp1-DNA interaction proposed earlier.


INTRODUCTION

Synthesis of mRNA by RNA polymerase II requires the interaction of a large array of auxiliary transcription factors that recognize and bind to specific promoter DNA sequences located upstream of eukaryotic genes (1, 2). These transcription factors regulate the initiation of transcription in a temporally ordered manner by assembling and engaging the active transcription complex. Consequently, many of the transcription factors have multiple domains responsible for sequence-specific DNA binding and transcriptional activation.

In order to understand the detailed roles played by each of the domains of sequence-specific transcription factors, efforts were made to fractionate the factors necessary to reconstitute transcriptional activity in vitro (3-5). These experiments resulted in the identification of one such promoter-specific transcription factor, Sp1, from HeLa cells (6-10). Sp1 enhances transcription from a variety of viral and cellular genes by binding to GC-rich decanucleotide recognition elements (GC boxes) within the 5'-flanking promoter sequences (10, 11). Although Sp1 can bind and activate transcription from a single GC box sequence (12), Sp1 binding sites often occur as multiple repeats (6, 9, 10, 13). However, Sp1 binds independently to each GC box sequence; physical interaction between adjacent Sp1 molecules is insufficient to give rise to cooperative DNA binding behavior (13). The multi-domain nature of Sp1 also facilitates Sp1-Sp1 interactions that occur in cases where Sp1 binding sites are widely separated (14-16). This self-association and DNA looping phenomenon are proposed to give rise to the observed transcriptional synergism or super-activation of Sp1 (14, 17).

The construction of truncated Sp1 fragments allowed the localization of the Zn2+-dependent DNA binding region to the carboxyl terminus, which was shown by sequence analysis to contain three "zinc finger" domains (7, 18-20). The Zn2+ domains found in Sp1 are analogous to those first identified in TFIIIA (21) and adopt the consensus sequence (FYH)XCX2-4CX3FX5LX2HX3HX5 (metal binding residues in bold) (22, 23). These domains are distinct from the Cys-rich motifs found in the steroid receptors (24), the yeast transcription factor GAL4 (25), or the Cys2-His-Cys motif observed in retroviral proteins (26). Structural modeling (27) and structural studies (28-35) show that Cys2-His2 domains contain two beta -strands with the Cys residues located at the beta -turn, and an alpha -helix containing the two His residues, oriented to coordinate Zn2+ in a tetrahedral fashion. This structural unit is now regarded as one of the major structural motifs involved in sequence-specific DNA binding and eukaryotic gene regulation.

Our general goal is to understand at the molecular level how the three zinc finger domains of Sp1 can bind with high affinity to a variety of GC box DNA sequences (10, 11). We have previously described the overexpression, purification, and characterization of a 92-amino acid peptide, Sp1-Zn92, that contains the three zinc fingers of Sp1 (36). The DNA binding properties of Sp1-Zn92 were surveyed using a variety of techniques based on gel electrophoresis to quantitatively analyze its interaction with several native and modified DNA sites. Sp1-Zn92 was shown to mimic the DNA binding properties of native Sp1 and, through comparisons with results from other zinc finger systems, a model was developed to explain the distinctive DNA binding properties of Sp1. Our model serves as the starting point for detailed studies of the conformations of the individual domains of Sp1-Zn92 aimed at defining those molecular features that allow Sp1 to recognize promoter sequences that contain the asymmetric GGGCGG hexanucleotide core (GC box) with a consensus sequence of 5'-(G/T)GGGCGG(G/A)(G/A)(G/T)-3'.

The distinguishing feature of Sp1-DNA binding is the high degree of sequence variability that is tolerated within the GC box with retention of high binding affinity (10, 11, 37). This raises interesting questions regarding detailed molecular mechanism of Sp1-DNA recognition process and the role which the individual fingers may have to play as "flexible-independent" reading domains in modulating binding to nonidentical DNA sites with near equal affinity. As a first step toward studying Sp1-DNA interactions, we have determined the solution structures of synthetic peptides corresponding to zinc finger domains two and three (N terminus to C terminus) of Sp1 using standard homonuclear NMR techniques. While zinc finger 3 belongs to the well defined Cys-X2-Cys structural subclass, zinc finger 2 is a member of the Cys-X4-Cys structural subclass, which has been defined for relatively few systems (32, 33, 35). The refined solution structures of zinc fingers 2 and 3 are compared with each other and with other reported zinc finger structures. Putative DNA binding residues are identified, and the individual roles of conserved residues are analyzed in the context of our previous model (36) of Sp1-DNA interactions.


EXPERIMENTAL PROCEDURES

Peptide Synthesis

Peptides corresponding to zinc finger domains 2 and 3 of Sp1 (Sp1f21 and Sp1f3, respectively, sequences shown in Fig. 3) were prepared at the Peptide Synthesis Facility, Keck Foundation Biotechnology Resource Laboratory, Yale University, using solid phase N-tert-butyloxycarbonyl chemistry. Amino acids were coupled by activated esters, and final deprotection/cleavage was done using hydrogen fluoride. Purification of the peptides was performed using Vydac C18 reverse phase columns and a linear gradient from 0.05% trifluoroacetic acid to 80% acetonitrile, 0.05% trifluoroacetic acid. The final product was lyophilized and characterized by analytical reverse phase high performance liquid chromatography, amino acid analysis, and laser desorption mass spectrometry. Peptide samples for all studies were stored under an argon atmosphere.


Fig. 3. Summary of sequential, short-range and medium range NOEs observed for Sp1f2 (top) and Sp1f3 (bottom) at 15 and 5 °C, respectively. The bars below the sequences represent the observed NOE connectivities. The thickness of the bars is a qualitative measure of cross-peak intensity in a 200-ms NOESY spectrum. Small (<6.0 Hz) and large (>8.0 Hz) values for Sp1f2 are indicated by filled and empty circles, respectively.
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Metal Binding Studies

The affinities of peptides Sp1f2 and Sp1f3 for Co2+ ion were determined using absorption spectroscopy (Perkin-Elmer Lambda 6 UV/VIS spectrophotometer) as a function of CoCl2 (99.999%, Aldrich) concentration in 50 mM HEPES, 50 mM NaCl, pH 8.0. All buffers were degassed using several freeze-pump-thaw cycles before use. The Co2+ dissociation constants were calculated using Equation 1 (38):
<FR><NU>l<SUB>0</SUB></NU><DE>&agr;</DE></FR>=<FR><NU>K<SUB>D</SUB></NU><DE>(1−&agr;)</DE></FR>+e<SUB>0</SUB> (Eq. 1)
where l0 = cobalt concentration in the buffer, alpha  = Delta /Delta max (Delta  = change in absorbance at 640 nm upon addition of cobalt and Delta max = maximum change in absorbance), KD = dissociation constant of cobalt-peptide complex, and e0 = total concentration of the Co2+ binding site (peptide concentration).

Circular Dichroism of Sp1f2 and Sp1f3

CD spectra of Sp1f2 and Sp1f3 (100 µg/ml peptide, 5 mM Tris·HCl, pH 8.0, 8 °C) were measured using an Aviv model 60DS spectropolarimeter. Spectra were recorded from 300 to 190 nm and averaged over five scans with bandwidth of 1.50 nm, scan step of 1.00 nm/point, and an averaging time of 10.0 s.

NMR Sample Preparation

Sp1f2 and Sp1f3 were dissolved in 0.5 ml of 25 mM Tris-d11 (Cambridge Isotope Laboratories), pH 7.5, containing 0.2% sodium azide (w/v) and 10% D2O (v/v) followed by the addition of ZnSO4 (20% molar excess). The final pH of the sample was adjusted to 5.90 (meter reading, uncorrected for isotope effect). The peptide concentrations were approximately 5 mM for each sample. All solutions were degassed by three freeze-pump-thaw cycles prior to protein dissolution, and all manipulations were carried out under an argon atmosphere. Samples were stored under an argon atmosphere and showed no degradation over the length of NMR experiments.

NMR Methods

NMR spectra were acquired using either a Bruker AM500 or a GE Omega500 NMR spectrometer. Two-dimensional NOESY2 spectra were acquired with selective water presaturation (delays alternating with nutations for tailored excitation pulse (39), AM500; Shinnar-LeRoux pulse (40), Omega500) followed by the standard NOESY pulse train (41). An inversion pulse bracketed by homospoil pulses was used during mixing time to minimize artifacts from the residual water resonance. Double quantum filtered correlation spectroscopy spectra were acquired with optimized phase cycling (42). Clean TOCSY spectra (43) were acquired using water saturation as given above, with MLEV17 (44) (AM500, Omega500) or decoupling in the presence of scalar interactions-2 (45) (Omega500) mixing schemes, followed by flipback and homospoil pulses for elimination of the rotating frame Overhauser effect and for water suppression. Quadrature detection in the indirect dimension was obtained using either time-proportional phase incrementation (AM500) or States-time-proportional phase incrementation (Omega500) (46, 47). Spectra were typically acquired with 32 or 48 scans per t1 value for 1024 t1 values, spectral width was typically 6000 Hz, and 2048 complex points were collected in the direct dimension. The free induction decay in both dimensions were multiplied by phase-shifted sine bell apodization function, zero-filled, and Fourier-transformed to yield 2048 by 2048 matrices. All spectra were processed using the FELIX 2.30 software package (Biosym, Inc.).

Structure Calculations

The hybrid distance geometry dynamical simulated annealing protocol within X-PLOR software package (48) was used for structure calculations. Interproton distances were calculated from cross-peak volumes derived from two-dimensional NOESY spectra recorded with a 200-ms mixing time, using a NH-NH (i, i + 1) distance of 2.8 Å as an internal standard. Upper and lower bounds were set equal to ±6% of the square of the distance calculated. For non-stereospecific assignments, distance constraints were applied to a pseudo atom situated at the geometric center of the nuclei, and the distance bounds were appropriately expanded, based on the known amino acid geometries. The experimental constraints were represented in the form of an asymmetric internuclear pseudo energy, having a minimum at the distance constraint, an infinite harmonic wall to the lower bound side, and a harmonic function making a transition to a zero slope asymptote to the upper bound side. The soft square potential function used in these calculations had a maximum potential of 50 kcal/mol, a soft square exponent of 2, and a scaling factor of 25.

3JHNHalpha coupling constants for Sp1f2 were determined from a double quantum filtered correlation spectroscopy spectrum using absorptive and dispersive antiphase splittings (49). The coupling constants were converted to torsional angle constraints using the Karplus relationship, and these restraints were used during the refinement step of Sp1f2 structure calculation protocol.

The structure calculations for Sp1f2 and Sp1f3 can be divided into three steps. In the first step, a set of substructures containing the backbone, Cbeta and Cgamma atoms were embedded in Cartesian coordinate space using the distance geometry protocol. Next, the remaining atoms were added in an extended conformation and subjected to multiple rounds of simulated annealing. Finally, the distance geometry simulated annealing regularized structures were subjected to multiple rounds of simulated annealing refinement. All calculations were performed using a SGI R4000 workstation.

Zinc Coordination

Initial structure calculations were performed without incorporating the Zn2+ atom; inspection of these structures clearly identified the Cys2-His2 Zn2+ binding ligands. Furthermore, analysis of the data allowed the unambiguous assignment of the Nepsilon atoms of His residues as the heteroatom coordinating the metal. Thereafter, Zn2+ was incorporated in structure calculations with an approximately tetrahedral geometry. Zinc-ligand bonds were assigned equilibrium distances of 2.30 Å [Zn-S] and 2.00 Å [Zn-N] (50) using artificial NOE constraints with a high weighing factor (300). The angles centered on the metal were constrained with tetrahedral equilibrium (harmonic potential). The Nepsilon atom of His ligands were constrained to lie in the plane defined by Cdelta 2, Cepsilon 1, and Zn2+ atoms.

Final structures were subjected to additional rounds of energy minimization, once after removing any lower bound on distance constraints and once without any explicit metal geometry constraints. Energy minimization without lower bounds had negligible effect on the average geometry of the calculated structures and did not increase the RMSD within the final structures for both Sp1f2 and Sp1f3. The energy minimization without metal binding constraints preserved the configuration and geometry of the Cys and His ligands around the metal binding site showing that metal-ligand constraints were consistent with the global energy minimum for the Sp1f2 and Sp1f3 structures.

The average coordinates and the RMSD values were calculated within X-PLOR, and the family of structures was visualized and overlaid using the software package Midas Plus Version 1.9. The structures with averaged coordinates (Sp1f2·avg, Sp1f3·avg) were subjected to a final round of simulated annealing refinement (refine·inp protocol of X-PLOR) to relieve bad contacts and irregular covalent geometry which might have arisen due to geometrical averaging. These average-refined structures (Sp1f2·avg, Sp1f3·avg) were used to identify potential hydrogen bonds and to model Sp1f2-DNA and Sp1f3-DNA interactions. The criteria for hydrogen bonds are that the distance between N of NH and O of CO (<UNL>N</UNL>H···<UNL>O</UNL>) be less than 3.4 Å and the angle N-O-C be larger than 110° (51).


RESULTS

Optical Studies of Sp1f2 and Sp1f3

The substitution of Co2+ for spectroscopically silent Zn2+ is a well established technique to probe the coordination environment of zinc containing metalloproteins (52-55). The visible absorption spectra (Fig. 1) associated with the d right-arrow d transitions of Co2+-substituted Sp1f2 and Sp1f3 show absorption maxima corresponding to two transitions centered around 640 nm (Sp1f2, epsilon M = 1080 M-1 cm-1) and 630 nm (Sp1f3, epsilon M = 1140 M-1 cm-1) with shoulders near 580 nm (Sp1f2, epsilon M = 504 M-1 cm-1) and 570 nm (Sp1f3, epsilon M = 511 M-1 cm-1). These absorption bands, which are responsible for the blue color of these metallopeptides, are eliminated in the presence of stoichiometric Zn2+, which readily displaces the coordinated Co2+ ions.


Fig. 1. Electronic absorption spectra of Co2+ bound to Sp1f2 (solid line) and Sp1f3 (dotted line) showing the Co2+-based d right-arrow d transitions.
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Optical titration experiments allowed the determination of Co2+ dissociation constants (KDCo) for Sp1f2 (KDCo Sp1f2 = 1.2 × 10-6) and Sp1f3 (KDCo Sp1f3 = 2.1 × 10-6). These KD values are consistent with values previously reported for zinc finger domains (62) and indicate a folded conformation for Sp1f2 and Sp1f3 in solution.

The positions and intensities of the d right-arrow d transitions are consistent with the formation of 1:1 Co2+-peptide complexes with the metal centers occupying tetrahedral or distorted tetrahedral environments for both Sp1f2 and Sp1f3 (54, 56, 57). Observation of the ligand field bands are analogous to those reported for Co2+ binding to the His2-Cys2 site in the second zinc finger domain of TFIIIA (56) and the gene 32 protein (58, 59). However, the definitive identification of coordination geometry based on UV/VIS spectroscopy is difficult owing to the similarity of the electronic spectra of distorted tetrahedral and five-coordinate Co2+ complexes, especially when only band positions are considered (53, 60, 61).

The circular dichroism spectra of both peptides show negative ellipticities at 228 nm, large negative molar ellipticities at 208 nm, and positive ellipticities at 190 nm (data not shown) in the presence of Zn2+. These features are consistent with the presence of regular secondary structure elements (63) and further indicate that the peptides adopt a conformation typical of folded zinc finger domains.

NMR Sequential Assignments and Secondary Structure Determination

Standard procedures that utilize two-dimensional COSY, TOCSY, and NOESY NMR data (64) were used to determine sequential resonance assignments (Figs. 2, A and B). The short and medium range connectivity patterns and the 3JHNHalpha coupling constants, summarized in Fig. 3, are consistent with an alpha -helical stretch for residues 17-28 (Sp1f2) and residues 16-27 (Sp1f3). Furthermore, the added presence of i, i + 2 connectivities indicates a 310 helical conformation for the last helical turn of both zinc finger domains. This transition from an alpha -helix to a 310 helix is a general property shared by the His-X3-His subclass of zinc fingers (structures with His-X4-5-His spacing show no indication of a 310 helix) (29). In both Sp1f2 and Sp1f3, the helix terminates with the Gly residue two residues after the second metal binding His. While the COOH-terminal portion of both peptides produced NOE patterns characteristic of a classical alpha -helix, no long uninterrupted connectivity patterns characteristic of a classical beta -strand were observed in either peptide. However, short segments preceding the first Cys residue and closely succeeding the second Cys residue gave strong Calpha Hi-NHi + 1 connectivities characteristic of an extended strand conformation. The observed NOE connectivities (Fig. 3) are also consistent with a turn among residues Trp7-Cys10 (Sp1f2) and residues Cys5-Cys8 (Sp1f3) connecting the two extended strands in each zinc finger domain.


Fig. 2. A, a portion of the TOCSY spectrum of Sp1f2 (mixing time 80 ms, 25 °C) showing backbone assignments. Boxes indicate the position of COSY cross-peaks. The Arg16 Nepsilon H-Cdelta H2 cross-peaks are marked with an asterisk. Note the unusual chemical shifts for the His27 beta -protons. B, a portion of the TOCSY spectrum of Sp1f3 (mixing time 60 ms, 5 °C) showing backbone assignments. The Arg14 Nepsilon H-Cdelta H cross-peaks are marked with asterisks.
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Three-dimensional Structures

The backbone conformations of Sp1f2 and Sp1f3 families are well defined by the NMR data, as shown in Fig. 4, A and B, and indicated by the average RMSDs for the peptides (Table I). As expected, the NH3+- and COO--terminal residues are poorly defined. This is reflected by the low RMSD of 0.43 Å for the backbone from the first conserved hydrophobic residue (Phe) through the residue immediately succeeding the second metal coordinating His residue.


Fig. 4. A, stereoview of the 20 energy minimized conformers used to represent solution structure of Sp1f2 (last residue not shown). The structures were overlaid to obtain the best match for the backbone for residues 3-27. All backbone heavy atoms including side chain heavy atoms of metal coordinating Cys and His residues are shown. B, stereoview of the 20 energy minimized conformers used to represent solution structure of Sp1f3 (last residue not shown). The structures were overlaid to obtain the best match for the backbone for residues 3-25. All backbone atoms including side chain heavy atoms of metal coordinating Cys and His residues are shown.
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Table I.

Analysis of the 20 calculated structures of Sp1f2 and Sp1f3 used to represent their solution conformation


Sp1f2 structures Sp1f3 structures

Based on 154 inter-residue distance constraints with six stereospecific beta -methylene assignments and 15 backbone torsion angle constraints Based on 180 inter-residue distance constraints with three beta -methylenes stereospecifically assigned
No NOE violation >0.3 Å outside the distance bounds and no dihedral angle violation >5° outside experimental bounds No NOE violation >0.3 Å outside the distance bounds
r.m.s difference of bond deviation from ideality <0.01 Å r.m.s difference of bond deviation from ideality <0.01 Å 
r.m.s difference bond angle deviations from ideality <2° r.m.s difference bond angle deviations from ideality <2°
Average r.m.s difference to the mean structure for non-H atoms = 2.32 Å Average r.m.s. difference to the mean structure for non-H atoms = 2.60 Å 
Average r.m.s difference to the mean structure for the backbone atoms = 1.57 Å Average r.m.s. difference to the mean structure for the backbone atoms = 1.76 Å 
Average r.m.s difference to the mean structure for non-H atoms from residues 3-28 = 1.24 Å Average r.m.s. difference to the mean structure for non-H atoms from residues 3-26 = 1.23 Å 
Average r.m.s difference to the mean structure for the backbone atoms from residues 3-28 = 0.43 Å Average r.m.s difference to the mean structure for the backbone atoms from residues 3-26 = 0.43 Å


DISCUSSION

The calculated structures exhibit secondary structure elements consistent with the short range NOE connectivity patterns. The overall topology of both peptides conforms to the expected fold of Cys2-His2 zinc finger domains consisting of two antiparallel strands linked by a Cys-Cys loop followed by a reverse 90° turn and an alpha -helix containing the two zinc coordinating His residues.

Observed long range NOEs define the relative orientation of the secondary structure elements. For Sp1f2 the NOE constraints are consistent with an antiparallel orientation for the two beta -strands extending from the turn encompassing Trp7-Cys10. Ninety percent of Sp1f2 structures are consistent with hydrogen bonds between Phe3(CO) and Arg14(NH) (distance <UNL>N</UNL>H···<UNL>O</UNL> = 2.9 Å, angle N-O-C = 163°) and between Cys5(NH) and Lys12(CO) (distance <UNL>N</UNL>H···<UNL>O</UNL> = 3.1 Å, angle N-O-C = 160°), stabilizing the antiparallel beta -sheet. The remainder of the amide and carbonyl groups in this sheet region are oriented as if interacting with the solvent. Long range connectivities in Sp1f3 also define an antiparallel orientation for its two beta -strands. The antiparallel beta -sheet of Sp1f3, however, seems to be much more open with no backbone-backbone hydrogen bonds evident in the average refined structure, as observed previously in the case of ADR1 and human enhancer proteins (30, 34).

Cys-Cys Loop

The results of NMR spectroscopy and Co2+ titration studies show that the increased size of the Cys-X4-Cys chelate of Sp1f2 does not dramatically alter either the local geometry or the metal affinity, relative to the more predominant Cys-X2-Cys subclass. There are, however, some differences in the turn conformations of the two zinc finger domains despite the fact that both turns require the two Cys residues to be positioned for tetrahedral metal binding. The Sp1f2 turn contains the residues Trp7 (i)-Ser8 (i + 1)-Tyr9 (i + 2)-Cys10 (i + 3), and the first metal coordinating Cys is completely excluded from the reverse turn (Fig. 5A). The turn is best classified as a beta -type II structural element based on the dihedral angle values observed for the average, refined structure (Table II). Hydrogen bonds are observed between Trp7(CO) and Cys10(NH) (distance <UNL>N</UNL>H···<UNL>O</UNL> = 3.73 Å, angle N-O-C = 109°) in 50% of the structures. The average refined structure, Sp1f2·avg·min, also shows the Sgamma of Cys10 within H-bonding distance of the backbone NH of Lys12 (distance <UNL>N</UNL>H···<UNL>S</UNL> = 3.4 Å, angle N-H-S = 124°). The turn conformation is further stabilized by the stacking of the Trp7 ring against the His27 imidazole ring (Fig. 6A). The orientation of the Trp indole ring with respect to His27 is borne out by the dramatic upfield shift for His27 beta -protons due to ring current effects (Fig. 2A).


Fig. 5. A, close-up view of family of 20 structures showing the Cys-X4-Cys region of Sp1f2. Predicted hydrogen bonds between Cys10 NH-Trp7 CO and Cys10 Sgamma -Lys12 NH are indicated by dotted lines. B, close-up view of the family of 20 structures showing the Cys-X2-Cys region of Sp1f3. Predicted hydrogen bonds between Cys5 NH-Cys8 CO, Cys5 Sgamma -Glu7 NH, and Cys5 Sgamma -Cys8 NH are indicated by dotted lines.
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Table II.

Dihedral angles

The dihedral angle values are for the average minimized structures of Sp1f2 and Sp1f3 (Sp1f2·avg and Sp1f3·avg, respectively). Residues i + 1 and i + 2 correspond to Pro6 and Glu7, respectively, for Sp1f3 and to Ser8 and Tyr9, respectively, for Sp1f2.
Turn type i + 1
i + 2 
 phi  psi  phi  psi

 beta -Type I  -60°  -30°  -90°
Sp1F3  -62°  -13°  -79°  -49°
 beta -Type II  -60° 120° 80°
Sp1F2  -86° 142° 76°


Fig. 6. A, the average, refined structure of Sp1f2 (Sp1f2·avg·min) showing orientations of well defined aromatic and apolar residues involved in hydrophobic packing (Phe3, Phe14, Leu20, Trp7), relatively well defined polar residues packing against the hydrophobic core via their alkyl methylene groups (Lys12, Glu19), long polar residues of the alpha -helix (Arg16, Asp18, Gln21, Arg22, Arg25), and the metal binding residues (Cys5, Cys10, His23, His27). B, the average, refined structure of Sp1f3 (Sp1f3·avg·min) showing orientations of well defined aromatic and apolar residues involved in hydrophobic packing (Phe3, Phe12, Leu18, Glu7), relatively well defined polar residue packing against the hydrophobic core via its alkyl methylene group (Lys10), long polar residues of the alpha -helix (Arg14, Asp16, Lys20, Lys23), and the metal binding residues (Cys5, Cys8, His21, His25).
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The Sp1f3 turn includes residues Cys5(i)-Pro6(i + 1)-Glu7 (i + 2)-Cys8 (i + 3) and is best classified as a beta -type I structural element, based on dihedral angle values (65) (Table II). The Sp1f3 beta -turn shows the expected hydrogen bond between CO of residue i (Cys5) and NH of residue i + 3 (Cys8) in all calculated structures (distance <UNL>N</UNL>H···<UNL>O</UNL> = 3.2 Å, angle N-O-C = 120°). In addition, the structures place the Sgamma of Cys5 within hydrogen bonding distance of the backbone NH groups of Glu7 (distance <UNL>N</UNL>H···<UNL>S</UNL> = 3.6 Å, angle N-H-S = 136°) and Cys8 (distance <UNL>N</UNL>H···<UNL>S</UNL> = 3.6 Å, angle N-H-S = 157°), thus further stabilizing the turn conformation (Fig. 5B). These NH···S hydrogen bond geometries are similar to those observed in ferrodoxin and rubredoxin (66). Also, the putative NH···S hydrogen bonds in Sp1f3 correspond to the sigma  and tau  bonds in the context of the SPXX motif (51), except that the Ser residue O atom is replaced by a Cys5 S atom. The psi  angle for the i + 2 Glu7 in Sp1f3 differs significantly from the expected value of 0° (Table II) (67), perhaps due to the fact that Glu7 is involved in a long range hydrophobic interaction with His23.

While it is not common for NH···S hydrogen bonds to occur in Cys residues involved in disulfide bridges, numerous NH···S bonds, as observed for Sp1f3, are a common feature in proteins coordinating metal ions (66). These bonds are hypothesized to play an important role in stabilizing the ligand arrangement required for metal coordination, thereby minimizing the entropy change caused by metal coordination to the apo protein.

Hydrophobic Core

Packing of the beta -sheet and alpha -helix of zinc finger domains against each other forms a hydrophobic core and places the conserved Cys and His residues toward the interior of the domain in a position to coordinate a Zn2+ ion. The experimental NOE constraints unambiguously determine the absolute chirality around the zinc ion as S, following earlier convention (27). Several residues (Phe14, Lys12, Trp7, and Lys24 for Sp1f2; Phe12, Lys10, Glu7, and Ile22 for Sp1f3) serve to shield the zinc ion from solvent and may therefore stabilize the metal-ligand interaction by precluding close approach of alternative donor ligands. The occurrence of such hydrophobic shells surrounding metal binding sites is well known and believed to play a key role by not only minimizing the change in conformational entropy upon metal binding by preordering the primary coordination sphere but also by precluding alternative modes of metal binding through the reduction of heteroatoms in the vicinity of the primary coordination sphere.

In addition to the expected packing interactions between aromatic and other hydrophobic side chains (Figs. 6, A and B and 7, A and B), alkyl methylene groups of certain long chain polar residues seem to be involved in hydrophobic interactions as well. For instance, in Sp1f2, the alkyl chain of Lys12 packs against the central Phe14 and His23, whereas the polar amine is oriented toward the solvent. Similarly, methylene groups of Glu19 and Lys24 (not visible) pack against Phe14 and His23, respectively, with their charged groups pointing outwards (Figs. 6A and 7A). A similar arrangement is seen for the side chain of Lys10 in Sp1f3 (Figs. 6B and 7B). Thus, despite their relatively small size, the zinc finger domains achieve a relatively high degree of packing and are stable as mini-globular domains in the presence of zinc and other divalent metal ions (Fig. 7, A and B).


Fig. 7. A, space-filling model of Sp1f2·avg·min with backbone atoms (dark gray), side chain carbons, and nitrogens of the metal binding and other hydrophobic residues (light gray), side chain carbons and nitrogens of long polar residues (white), side chain hydrogens (white), and zinc atom (black) illustrating the well packed mini-globular nature of the zinc finger domain. Note how the putative DNA binding residues Arg16 (-1) and Arg22 (+6) point toward the exterior, whereas the side chain of Lys12, Glu19 pack in with Phe14, and Gln21 is folded in along the backbone. Lys12 and His23 are well positioned to make proposed DNA phosphate contacts via their exposed nitrogen atoms3 (marked with asterisks). B, space-filling model of Sp1f3·avg·min (same coloring scheme as Fig. 9A) illustrating the well packed mini-globular nature of the zinc finger domain. Putative DNA binding residues are Arg14 (-1) and His17 (+3). Lys10 and His21 are proposed to contact the DNA backbone via their exposed nitrogen atoms (marked with asterisks).
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Sp1-DNA Interactions

In the Zif268·DNA complex crystal structure the zinc fingers bind DNA by docking their alpha -helices in the major groove such that each zinc finger makes contact with the G-rich strand of the appropriate cognate 3-base pair subsite using residues at positions -1, +3, or +6 relative to the start of the alpha -helix (32). Fingers 1 and 3 of Zif268 use Arg residues -1 and +6 to contact the underlined residues of the <UNL><B>G</B></UNL>C<UNL><B>G</B></UNL> subsite, and finger 2 uses residues Arg (-1) and His (+3) to contact the underlined bases of G<UNL><B>GG</B></UNL> subsite. An analysis of the Sp1 sequence, considering the residues analogous to the ones involved in DNA binding in the Zif268 structure, reveals striking similarities between the zinc fingers of Sp1 and Zif268 (36). In light of these similarities, we proposed a model for interaction of Sp1 with DNA based on the Zif268/DNA co-crystal structure which envisaged Sp1f2, by analogy to Zif268 finger 1, using Arg (-1) (residue 16) and Arg (+6) (residue 22) for <UNL><B>G</B></UNL>C<UNL><B>G</B></UNL> recognition and Sp1f3, by analogy to Zif268 finger 2, using Arg (-1) (residue 14) and His (+3) (residue 17) for G<UNL><B>GG</B></UNL> recognition (Fig. 8) (36, 68).


Fig. 8. Summary of proposed DNA contacts between Sp1f2 and Sp1f3 with their respective 3-base pair subsites. The proposed amino acid-DNA base contacts are shown by a solid line. The other probable amino acid-DNA base interactions within the framework are indicated by dotted lines. Note the antiparallel orientation of peptide and DNA and absence of any direct contacts with the other C-rich strand of DNA duplex (not shown).
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To test the validity of the above-mentioned model and to gain further insight into the role of individual amino acid residues, we superimposed the average refined structures of Sp1f2 and Sp1f3 on Zif268 fingers 1 and 2 in the Zif268-DNA crystal structure (69). As required by the model, the structures of Sp1f2 and Sp1f3 were found to be very similar to Zif268 fingers 1 and 2, respectively (Fig. 9, A and B). The backbone RMSD values for residues 3-25 of Sp1f3 (excluding the residue immediately succeeding the second metal binding Cys) and the corresponding 22 residues of Zif268 finger 2 is 0.90 Å. The backbones deviate significantly at the residue succeeding the second Cys, due to the difference in phi  angles at this position. Sp1f3 contains a Pro residue at this site that has its phi  angle restricted to -60°, whereas most zinc fingers, including Zif268 finger 2, exhibit a positive phi angle for the residue immediately succeeding the second Cys location (69). The best overlay of Sp1f2 and Zif-268 finger 1 between the first conserved hydrophobic residue to the second His residue (excluding the four residues in the Cys-Cys loop) gives an RMSD value of only 0.68 Å. The residues of the Cys-X4-Cys loop were excluded from the RMSD calculation since these residues were stated to be ill-defined in the crystal structure (32).


Fig. 9. A, stereo presentation of the superposition of Sp1f2·avg·min (see "Experimental Procedures") and Zif268 finger 1. Residues 3-27 (excluding the loop between the two Cys residues) of Sp1f2·min·avg (dark) were superimposed on the corresponding residues of Zif268 finger 1 (light). B, stereo presentation of the superposition of Sp1f3·avg·min (see "Experimental Procedures") and Zif268 finger 2. Residues 3-25 (excluding Pro9) of Sp1f3·min·avg (dark) were superimposed on the corresponding residues of Zif268 finger 2 (light).
[View Larger Version of this Image (38K GIF file)]


Overlay of Sp1f2 with Zif-268 finger 1 in the co-crystal structure almost exactly overlaps the backbone atoms of their respective alpha -helices, and even though the side chains of residues -1 (Arg16) and +6 (Arg22) of Sp1f2 are not constrained in the NMR structures, their backbone alpha  carbons are positioned such that the side chains start out pointing toward the major groove of DNA (Fig. 9A) in a manner that is consistent with their proposed interaction with DNA bases, which would be the underlined guanines of the <UNL>G</UNL>C<UNL>G</UNL> subsite if we assume that Sp1f2 docks in the major groove in an orientation similar to that observed for Zif268 finger 1 in the crystal structure.3 This mode of interaction is also consistent with the protection/interference data (70) and the complete conservation of the underlined guanines of the subsite <UNL><B>G</B></UNL>C<UNL><B>G</B></UNL> in all Sp1 sites identified to date (10). In contrast to Arg (-1) and Arg (+6), Glu (+3) (residue 19) of Sp1f2, whose side chain is relatively well defined in the NMR structures (average RMSD of side chain carbon atoms = 1.07 Å), does not point toward the major groove but instead packs its methylene beta -protons against the central Phe in a manner similar to the Zif268 Glu (+3) residue (Figs. 6A and 7A), suggesting that it may not interact directly with DNA. In fact when all the 20 structures of Sp1f2 were overlaid on Zif268 finger 1, as described before, the Glu (+3) side chain did not come within interacting distance of any base of DNA in 19 of the 20 models generated. This is consistent with the reported absence of methylation protection of the middle C position of the 3-base pair <UNL><B>G</B></UNL>C<UNL><B>G</B></UNL> subsite. Gln (+5) (residue 21) is another hydrophilic residue in the alpha -helix that is relatively well defined in the NMR structures (average RMSD of side chain carbon atoms = 1.08 Å) and does not point toward the major groove. This residue, instead, folds back along the backbone (Figs. 6A and 7A) and appears to place its side chain amide proton within hydrogen bonding distance of Ser17 side chain Ogamma and carbonyl O atoms, although it is difficult to clearly identify the interacting partner. Thus we see that the model generated by the overlay and the side chain packing arrangement is consistent with Arg16 and Arg22 interacting with DNA bases but tends to preclude the possibility of the other two polar residues on the alpha -helix (Glu19 and Gln21) being involved in direct base recognition.

Similar overlay of Sp1f3 with Zif268 finger 2 (Fig. 9B) positions Arg (-1) (residue 14) and His (+3) pointing (residue 17) toward the major groove consistent with proposed DNA contacts. The Sp1f3/DNA model, however, does not preclude the interaction of Lys (+6) (residue 20) with DNA. The corresponding residue in Zif268 finger 2 is a Thr which is too short to contact DNA. Protection/interference data for Sp1 indicate that the guanine base expected to be contacted by Lys (+6) (<UNL><B>G</B></UNL>GG) is only weakly interacting and can be replaced by a thymine residue. The reason for this apparently weak interaction is not clear and may be entirely due to the side chain length of Lys being incompatible with the shorter DNA binding His at +3 position for making simultaneous DNA contacts (71).

Arg-Asp Interaction

The oxygens of the carboxylate group of Asp (+2) were found to be in a hydrogen bond-salt bridge interaction with Nepsilon group of Arg (-1) in all the three zinc fingers of Zif268 crystal structure. There is NMR spectral evidence suggesting that a similar interaction exists between the analogous side chains Arg16-Asp18 of Sp1f2 and Arg14-Asp16 of Sp1f3 even in the absence of DNA (69). In Sp1f3 the Nepsilon H proton of Arg14 gives intense TOCSY cross-peaks with neighboring protons in the side chain and is shifted downfield to 8.02 ppm from the random-coil value of 7.20 ppm (Fig. 2B) suggesting that the Arg14 Nepsilon H proton is protected from exchange and probably involved in a hydrogen bond. Arg14 is also the only long hydrophilic side chain to show a large chemical difference between the two diastereotopic methylene protons of the terminal CH2 group (Delta ppm (Cdelta H2) = 0.50 at 5 °C) (Fig. 2B). This large chemical shift difference indicates a well defined solution conformation for Arg14 side chain. Furthermore, inspection of the NMR spectra revealed a moderately strong NOE between the Cgamma H proton of Arg14 and Cbeta H proton of Asp16 (this NOE was not included in the structure calculations). The above-mentioned facts taken together strongly suggest that the Arg14 Nepsilon H proton is involved in a hydrogen bond-like interaction, in all probability with the side chain of Asp16, even though this interaction is not apparent in all the individual structures due to lack of NOE constraints. The Asp18 of Sp1f2 is also capable of having a similar interaction with Arg16. Again the terminal Nepsilon H of Arg16 (Delta ppm (Cdelta H2) = 0.06 at 15 °C) (Fig. 2A) gives an intense and considerably downfield shifted resonance in the NMR spectra indicating that Arg-Asp interaction also exists in Sp1f2 free in solution unbound to DNA. This Arg-Asp interaction is presumed to stabilize the long side chain of Arg and enhance the specificity of arginine-guanine interaction. The presence of this interaction in Sp1f2 and Sp1f3 further implicates Arg (-1) of both domains in DNA binding.

We have also acquired NMR spectra and obtained backbone assignments of an over-expressed peptide fragment containing both the zinc finger domains 2 and 3 (Sp1f23). Sp1f23 then represents two-thirds of the DNA binding domain of Sp1 and has been shown to be capable of binding DNA in a Zn2+-dependent, sequence-specific manner.4 We found that for most part the NMR spectrum of Sp1f23 construct is close to the sum of the NMR spectra of Sp1f2 and Sp1f3 (except for the residues in the linker between the two domains), indicating negligible domain-domain interactions while free in solution. Since chemical shifts are very sensitive to local structure, this further supports the idea that the single finger structures are very relevant in context of larger domains and can serve as reasonable models to understand the mode of sequence-specific DNA interaction of entire multifinger constructs.

Furthermore, this essentially allows the transfer of Sp1f2 and Sp1f3 assignments to the larger Sp1f23 fragment, thereby greatly facilitating assignment of the entire DNA binding domain of Sp1 in a modular fashion.

Conclusions

It is our aim to understand the chemical basis of the unique Sp1 DNA recognition process at the molecular level. Towards this objective, we have solved the solution structures of synthetic peptides corresponding to zinc finger domains 2 and 3 of Sp1, using homonuclear two-dimensional NMR spectroscopy. Circular dichroism studies and Co2+ titration experiments show that both peptides assume a folded conformation around a tetrahedral metal center with no significant differences in the metal binding affinities between the Cys-X4-Cys (Sp1f2) and Cys-X2-Cys (Sp1f3) subclasses. Sp1f2 has a stable beta -type I turn between the two strands with the first Cys residue excluded from the turn motif due to the longer -X4- loop. Sp1f3 contains the sequence Cys-Pro-Glu-Cys-Pro in which the first Pro causes the turn to closely resemble the SPXX motif both in geometry and hydrogen bonding pattern. The second Pro forces the phi  angle at that position to be fixed at about -60 degrees, contrary to the expected positive value at this position. This may be the reason for the antiparallel beta -sheet being more open in Sp1f3. The NMR solution structures show several relatively well defined polar side chains making hydrophobic contacts with the central apolar residues via their alkyl methylene groups or aromatic rings while pointing their charged atoms away toward the solution. Such residues include Lys12 and His23 of Sp1f2 and Lys10 and His21 of Sp1f3. It is interesting to note that polar groups of residues corresponding to these very positions show interactions with DNA backbone phosphates in the Zif268/DNA co-crystal structure. Since these side chains seem to have a relatively fixed orientation even free in solution, these interactions could play an important role in correctly docking and orienting the zinc fingers in the major groove of DNA.

The comparison of NMR spectra of Sp1f23 with those of Sp1f2 and Sp1f3 supports the idea that zinc fingers fold as independent, noninteracting entities with structures very relevant in the context of the entire protein bound to DNA. The free solution structures of Sp1f2 and Sp1f3 are very similar to those of analogous zinc finger domains of Zif268 bound to DNA. Modeling Sp1-DNA complex by overlaying the Sp1f2 and Sp1f3 structures on Zif268 fingers 1 and 2, respectively, predicts the role of key amino acid residues, the interference/protection data, and is consistent with the model of Sp1-DNA interaction proposed earlier. Interestingly, the Arg-Asp buttressing interaction observed in Zif268/DNA crystal structure also seems to be preserved in Sp1f2 and Sp1f3, free in solution. The presence of this interaction in single zinc fingers without DNA further strengthens the emerging theme that zinc fingers are preformed, prearranged motifs, ready to interact with DNA even at the level of individual subdomains. Thus we expect only a very small entropic cost to be associated with sequence-specific recognition of DNA by Sp1 zinc fingers two and three.

In conclusion, the structures of Sp1f2 and Sp1f3 presented above are important steps toward understanding the DNA binding domain of Sp1. These data offer insight into both the structural features of zinc fingers and the mechanisms of sequence-specific interaction of Sp1 with DNA. Work is in progress using both mutagenesis and NMR spectroscopy to further characterize and understand the Sp1-DNA recognition process and define the chemical basis of the unique features of Sp1 binding.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant GM 48346 and in part by the Alfred P. Sloan Foundation (Research Fellow, J. P. C.).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.

The coordinates of the average refined structures have been deposited in the Brookhaven Protein Data Bank under the file names Sp1f2·avg and Sp1f3·avg.


   Supported in part by a National Institutes of Health Predoctoral Biophysical Fellowship 5 T32 GM08293-04.
§   Current address: Dept. of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.
   To whom correspondence should be addressed: Dept. of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107.
1   A table of chemical shifts of Sp1f2 and Sp1f3, solution structure families of Sp1f2 and Sp1f3 showing key side chains, input files used for XPLOR calculations, and plots used to calculate cobalt dissociation constants for Sp1f2 and Sp1f3 are available on request. Ordering information is given on any current masthead page.
2   The abbreviations used are: NOESY, nuclear Overhauser effect spectroscopy; NOE, nuclear Overhauser effect; RMSD, root mean square deviation; rotating frame Overhauser effect TOCSY, total correlation spectroscopy.
3   Overlay of Sp1f2·avg structure on Zif268 finger 1 places the terminal NH protons of the relatively well defined Lys12 (average RMSD of side chain carbons until Cdelta  = 0.75 Å) within hydrogen bonding distance of 5'-phosphate of base pair 7, analogous to Zif268 structure (Figs. 6A and 9A). Additionally, overlays of both Sp1f2 and Sp1f3 with Zif fingers 1 and 2, respectively, in the Zif268-DNA complex place the Nepsilon proton of the first metal coordinating His within hydrogen bonding distance of the 5'-phosphate of base pairs 4 and 7, respectively, as observed for the corresponding positions in the Zif268 complex (Fig. 9, A and B). The preservation of these contacts with DNA backbone (which have been proposed to serve to precisely position the alpha -helix with respect to their DNA sites in the major groove (32)), upon superpositioning of the structures suggests that the orientation and docking mode of Sp1f2 and Sp1f3 in the major groove of DNA must be very similar to that observed for the analogous Zif268 zinc fingers.
4   X. Cao, unpublished results.

Acknowledgments

We thank Prof. James P. Prestegard for useful comments and discussions, Ranajeet Ghose for helpful advice with NMR experiments, and Xiaohong Cao for providing us with purified Sp1f23.


REFERENCES

  1. Lewis, M. K., and Burgess, R. R. (1982) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 15B, pp. 109-153, Academic Press, New York
  2. Johnson, P. F., and McKnight, S. L. (1989) Annu. Rev. Biochem. 58, 799-839 [CrossRef][Medline] [Order article via Infotrieve]
  3. Kadonaga, J. T., and Tjian, R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5889-5893 [Abstract]
  4. Ptashne, M. (1988) Nature 335, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  5. Schöler, H. J., Hatzopoulos, A. K., and Schlokat, U. (1988) in Architecture of Eukaryotic Genes (Kahl, G., ed), pp. 89-122, VCH, Frankfurt
  6. Dynan, W. S., and Tjian, R. (1983) Cell 32, 669-680 [Medline] [Order article via Infotrieve]
  7. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, 1079-1090 [Medline] [Order article via Infotrieve]
  8. Dynan, W. S., and Tjian, R. (1983) Cell 35, 79-87 [Medline] [Order article via Infotrieve]
  9. Gidoni, D., Dynan, W. S., and Tjian, R. (1984) Nature 312, 409-413 [Medline] [Order article via Infotrieve]
  10. Kadonaga, J. T., Jones, K., and Tjian, R. (1986) Trends Biochem. Sci. 11, 201-203
  11. Bucher, P. (1990) J. Mol. Biol. 212, 563-578 [Medline] [Order article via Infotrieve]
  12. Letovsky, J., and Dynan, W. S. (1989) Nucleic Acids Res. 17, 2639-2653 [Abstract]
  13. Gidoni, D., Kadonaga, J. T., Barrera-Saldana, H., Takahashi, K., Chambon, P., and Tjian, R. (1985) Science 230, 511-517 [Medline] [Order article via Infotrieve]
  14. Mastrangelo, I. A., Courey, A. J., Wall, J. S., Jackson, S. P., and Hough, P. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5670-5674 [Abstract]
  15. Pascal, E., and Tjian, R. (1991) Genes Dev. 5, 1646-1656 [Abstract]
  16. Courey, A. J., and Tjian, R. (1988) Cell 55, 887-898 [Medline] [Order article via Infotrieve]
  17. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Cell 59, 827-836 [Medline] [Order article via Infotrieve]
  18. Briggs, M. R., Kadonaga, J. T., Bell, S. P., and Tjian, R. (1986) Science 234, 47-52 [Medline] [Order article via Infotrieve]
  19. Westin, G., and Schaffner, W. (1988) Nucleic Acids Res. 16, 5771-5781 [Abstract]
  20. Kadonaga, J. T., Courey, A. J., Ladika, J., and Tjian, R. (1988) Science 242, 1566-1570 [Medline] [Order article via Infotrieve]
  21. Miller, J., McLachlan, A. D., and Klug, A. (1985) EMBO J. 4, 1609-1614 [Abstract]
  22. Jacobs, G. H. (1992) EMBO J. 11, 4507-4517 [Abstract]
  23. Brown, R. S., Sander, C., and Argos, P. (1985) FEBS Lett. 186, 271-274 [CrossRef][Medline] [Order article via Infotrieve]
  24. Omichinski, J. G., Clore, G. M., Appella, E., Sakaguchi, K., and Grönenborn, A. M. (1990) Biochemistry 29, 9324-9339 [Medline] [Order article via Infotrieve]
  25. Pan, T., and Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2077-2081 [Abstract]
  26. Summers, M. F., South, T. L., Kim, B., and Hare, D. R. (1990) Biochemistry 29, 329-340 [Medline] [Order article via Infotrieve]
  27. Berg, J. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 99-102 [Abstract]
  28. Parraga, G., Horvath, S. J., Eisen, A., Taylor, W. E., Hood, L., Young, E. T., and Klevit, R. E. (1988) Science 241, 1489-1492 [Medline] [Order article via Infotrieve]
  29. Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A., and Wright, P. E. (1989) Science 245, 635-637 [Medline] [Order article via Infotrieve]
  30. Klevit, R. E., Herriott, J. R., and Horvath, S. J. (1990) Proteins 7, 215-226 [Medline] [Order article via Infotrieve]
  31. Kochoyan, M., Havel, T. F., Nguyen, D. T., Dahl, C. E., Keutmann, H. T., and Weiss, M. A. (1991) Biochemistry 30, 3371-3386 [Medline] [Order article via Infotrieve]
  32. Pavletich, N. P., and Pabo, C. O. (1991) Science 252, 809-817 [Medline] [Order article via Infotrieve]
  33. Neuhaus, D., Nakaseko, Y., Schwabe, J. W., and Klug, A. (1992) J. Mol. Biol. 228, 637-651 [Medline] [Order article via Infotrieve]
  34. Omichinski, J. G., Clore, G. M., Robien, M., Sakaguchi, K., Appella, E., and Gronenborn, A. M. (1992) Biochemistry 31, 3907-3917 [Medline] [Order article via Infotrieve]
  35. Pavletich, N. P., and Pabo, C. O. (1993) Science 261, 1701-1707 [Medline] [Order article via Infotrieve]
  36. Kriwacki, R. W., Schultz, S. C., Steitz, T. A., and Caradonna, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9759-9763 [Abstract]
  37. Jones, K. A., Kadonaga, J. T., Luciw, P. A., and Tjian, R. (1986) Science 232, 755-759 [Medline] [Order article via Infotrieve]
  38. Bagshaw, C. R., and Harris, D. A. (1987) in Spectrophotometry and Spectrofluorimetry: A Practical Approach (Harns, D. A., and Bashford, C. L., eds), pp. 91-113, IRL Press, Washington, D. C.
  39. Morris, A. G., and Freeman, R. (1978) J. Magn. Reson. 29, 433-462
  40. Shinnar, M., Elff, Subramanian, H., and Hurd, R. E. (1989) Magn. Reson. Med. 12, 75-79
  41. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4533-4546
  42. Derome, A. E., and Williamson, M. P. (1990) J. Magn. Reson. 88, 177-185
  43. Griesenger, C., Otting, G., Wüthrich, K., and Ernst, R. R. (1988) J. Am. Chem. Soc. 110, 7870-7872
  44. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355-360
  45. Shaka, A. J., Lee, C. J., and Pines, A. (1988) J. Magn. Reson. 77, 274-293
  46. Bodenhausen, G., Vold, R. I., and Vold, R. R. (1980) J. Magn. Reson. 37, 93-106
  47. Marion, D., and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 967-974 [Medline] [Order article via Infotrieve]
  48. Brunger, A. T. (1990) X-PLOR Manual, version 3.1, Yale University Press, New Haven
  49. Kim, Y., and Prestegard, J. H. (1989) J. Magn. Reson. 84, 9-13
  50. Diakun, G. P., Fairall, L., and Klug, A. (1986) Nature 324, 698-699 [Medline] [Order article via Infotrieve]
  51. Suzuki, M. (1991) Proc. R. Soc. Lond. B Biol. Sci. 246, 231-235 [Medline] [Order article via Infotrieve]
  52. Chlebowski, J. F., and Coleman, J. E. (1976) Metal Ions Biol. Syst. 6, 1-140
  53. Gray, H. B. (1980) in Methods for Determining Metal Ion Environments in Proteins (Darnall, D. W., and Wilkins, R. G., eds), Vol. 2, pp. 1-26, Elsevier Science Publishing Co., Inc., New York
  54. Bertini, I., and Luchinat, C. (1984) Vol. 6, pp. 71-111, Elsevier Science Publishing Co., Inc., New York
  55. Maret, W., and Vallee, B. L. (1993) Methods Enzymol. 226, 52-71 [Medline] [Order article via Infotrieve]
  56. Frankel, A. D., Berg, J. M., and Pabo, C. O. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4841-4845 [Abstract]
  57. Green, L. M., and Berg, J. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4047-4051 [Abstract]
  58. Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., and Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8452-8456 [Abstract]
  59. Qiu, H., and Giedroc, D. P. (1994) Biochemistry 33, 8139-8148 [Medline] [Order article via Infotrieve]
  60. Rosenberg, R. C., Root, C. A., and Gray, H. B. (1975) J. Am. Chem. Soc. 97, 21-26 [Medline] [Order article via Infotrieve]
  61. Lions, F., Dance, I. G., and Lewis, J. (1967) J. Chem. Soc. 4, 565-572
  62. Berg, J. M., and Merkle, D. L. (1989) J. Am. Chem. Soc. 111, 3759-3760
  63. Johnson, W. C. J. (1990) Proteins Struct. Funct. Genet. 7, 205-214 [Medline] [Order article via Infotrieve]
  64. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley Interscience, New York
  65. Rose, G. D., Gierasch, L. M., and Smith, J. A. (1985) Adv. Protein Chem. 37, 1-109 [Medline] [Order article via Infotrieve]
  66. Adman, E., Watenpaugh, K. G., and Jensen, H. C. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 44854-4858
  67. Hutchinson, G. E., and Thornton, J. M. (1994) Protein Sci. 3, 2207-2216 [Abstract/Free Full Text]
  68. Berg, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11109-11110 [Free Full Text]
  69. Bernstein, B. E., Hoffman, R. C., Horvath, S., Herriott, J. R., and Klevit, R. E. (1994) Biochemistry 33, 4460-4470 [Medline] [Order article via Infotrieve]
  70. Kuwahara, J., Yonezawa, J., Futamura, A., and Sugiyura, Y. (1993) Biochemistry 32, 5994-6001 [Medline] [Order article via Infotrieve]
  71. Desjarlais, J. R., and Berg, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7345-7349 [Abstract]

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