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Originally published In Press as doi:10.1074/jbc.M703772200 on December 11, 2007

J. Biol. Chem., Vol. 283, Issue 8, 5158-5167, February 22, 2008
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Structural and Biophysical Analysis of the DNA Binding Properties of Myelin Transcription Factor 1*

Roland Gamsjaeger12, Michael K. Swanton13, Felix J. Kobus, Eija Lehtomaki, Jason A. Lowry, Ann H. Kwan, Jacqueline M. Matthews4, and Joel P. Mackay5

From the School of Molecular and Microbial Biosciences, University of Sydney, Building G08, New South Wales, Sydney 2006, Australia

Received for publication, May 8, 2007 , and in revised form, November 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is predicted that there are at least 15,000 zinc fingers (ZnFs)6 in over 1,000 different proteins in humans (1). These domains fall into more than twenty subclasses, based on their fold and zinc-ligation topology, and different members can mediate interactions with DNA, RNA, proteins, and other molecules (2). The structures and functions of several classes of ZnFs, including classical and GATA-type domains, have been analyzed in detail; in contrast, some classes of ZnFs are not well understood.

Myelin transcription factor 1 (MyT1, or neural zinc finger 2 (NZF2)) is a transcription factor that contains seven copies of a ZnF with a C2HC consensus sequence. The seven ZnFs are arranged in a 1 + 2 + 4 topology (see Fig. 1), although a second isoform exists that lacks finger 1 (F1) (3). MyT1 was first isolated on the basis of its interaction with DNA elements in the promoter of the proteolipid protein (PLP) gene (4). MyT1 acts during central nervous system development to induce differentiation of early neuronal precursors and to commit glial cell precursors to the glial cell lineage (5). After birth, MyT1 activates the PLP gene in Schwann cells, leading to the production of the myelin sheath around axons in the central nervous system (6, 7). Orthologs of MyT1 are found in organisms ranging from nematodes to mammals, and functional data indicate that MyT1 is essential for neural development in Xenopus laevis (5). Humans contain two other likely paralogs of MyT1: MyT1-like (MyT1L/NZF1) and Suppressor of Tumorigenicity 18 (ST18/NZF3). MyT1L appears to be involved in neural development (7), whereas ST18 was reported to be a breast cancer tumor suppressor (8). At least three additional vertebrate proteins that contain one MyT1-type ZnF have been identified (L3MBTL, L3MBTL3, and L3MBTL4), and one of these, L3MBTL, is a possible tumor suppressor (9).

Several biochemical studies have demonstrated that MyT1-type ZnFs from MyT1, MyT1L, and ST18 can recognize DNA in a sequence-specific manner (5, 10, 11). The consensus sequence, AAGTT, is the core element of the cis-regulatory element of the β-retinoic acid receptor gene (β-RARE) and is also found in the human PLP promoter. Until now, the molecular details of DNA recognition by MyT1-type zinc fingers have not been addressed.

In this work, we used surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to delineate the relative importance of each ZnF in MyT1 for DNA binding. Finger 5 (F5) has the highest affinity for the β-RARE site. We went on to use a combination of NMR and mutagenesis to probe the structural basis for the MyT1-DNA interaction. A data-driven model of the interaction, calculated using HADDOCK (12-14), reveals that a whole MyT1-type ZnF can sit snugly in the major groove of double-stranded DNA, forming a number of specific electrostatic and hydrophobic contacts. This mode of recognition is distinct from that observed for other classes of DNA-binding ZnFs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Subcloning, Expression, and Purification of MyT1 Constructs—The original plasmid encoding mouse 6-ZnF myelin transcription factor 1 (mMyT1) was a generous gift of Dr. Lynn Hudson (National Institutes of Health). Ten different constructs of MyT1 were cloned from the original plasmid and mutants were constructed using overlap extension PCR (see Fig. 1). All constructs were overexpressed as fusions with glutathione S-transferase at 25 °C under standard conditions; isotopically labeled F5 was overexpressed using the protocol described in a previous study (15). Proteins were purified using GSH affinity chromatography, thrombin cleavage, and gel filtration (Superdex-75 in SPR buffer: 50 mM NaCl, 10 mM HEPES, 1 mM dithiothreitol, pH 7.4). Protein identities were confirmed by electrospray mass spectrometry, and concentrations were determined by absorbance at 215, 225, and 280 nm (16). Fractions were stored at -20 °C until required.

β-RARE DNA and Mutant Oligonucleotides—Single-stranded β-RARE DNA (5'-ACCGAAAGTTCAC and 5'-GTGAACTTTCGGT), mutant oligonucleotides and biotinylated DNA for SPR experiments were obtained from Sigma-Aldrich, annealed in SPR buffer without dithiothreitol (heated to 95 °C for 5 min, then cooled to room temperature over the course of 1-2 h) and purified using gel filtration (Superdex-75). Concentrations were calculated by absorbance at 260 nm.

SPR—All experiments were performed on a Biacore 3000 system (Biacore AB) at flow rates of 5-10 µl/min in SPR buffer. Biotinylated DNA (~10-100 nM) was immobilized on streptavidin-coated Biacore SA chips. MyT1 (1-110 µM) was injected in SPR buffer and binding was monitored. To compare binding affinities for different single- or double-finger constructs in a semi-quantitative manner, equal concentrations of each construct were injected (10 or 20 µM) and the SPR responses were compared. The system was washed with 1 M NaCl (1 min) after each experiment. For kinetics studies (F5 and F7), curves were fitted using the standard 1:1 Langmuir model. In the competition experiments, either F5 (12 µM) or F45 (5 µM) was added to pre-bound DNA in the presence of 2 or 5 molar equivalents of competitor DNA oligonucleotides, respectively.

ITC—Protein and DNA samples were dialyzed overnight against the same reservoir of SPR buffer. β-RARE DNA (60-130 µM) was titrated into F5 or F45 (8 µM). Titrations were carried out on a MicroCal VP-ITC microcalorimeter at 25 °C. For each titration, 25-30 injections of 5-10 µl of titrant were made at 5-min intervals. Data were corrected for heats of dilution from control experiments of DNA into buffer, and analyzed using Origin ITC Analysis software (MicroCal Software, Northampton, MA), as described previously (17).

NMR Spectroscopy—F5 (unlabeled, 15N- or 15N/13C-labeled) was dialyzed into NMR buffer (2.5 mM deuterated MES, pH 6.5, 10 mM NaCl, 1 mM dithiothreitol or Tris(2-chloroethyl)phosphite) and concentrated in Microsep 1-K cutoff units to 200-700 µM. Standard homonuclear and 15N-separated experiments were acquired at 4 °C on a Bruker Avance 600 and Avance 800 NMR spectrometers equipped with cryoprobes. 15N-separated NOESY and two-dimensional NOESY data were used to derive distance constraints. Dihedral restraints were obtained by analysis of the HNHA spectrum (18), and stereo-specific assignments were made from short mixing time NOESY (50 ms) and TOCSY (35 ms) data. 15N-HSQC titrations of F5 with β-RARE DNA were carried out in 10 mM sodium phosphate buffer with 50 mM NaCl (pH 7.4) in the presence of 1 mM dithiothreitol at 25 °C. HNCA, HNCACB, CBCA(CO)NH, and HNCO experiments were recorded for both 15N,13C-F5 alone, and the protein·DNA complex, together with single and double half-filtered NOESY experiments and an HCCH-TOCSY for the complex. Chemical shift changes were calculated as a weighted average of HN,N,C{alpha}, and C' changes, using to a previously reported equation (19, 20). Assignments of the DNA alone were obtained from two-dimensional NOESY spectra. NMR data were processed using XWINNMR (Bruker, Karlsruhe) and analyzed with SPARKY (T. D. Goddard and D. G. Kneller, University of California at San Francisco).

Structure Calculations—Initial structures of F5 were calculated in DYANA (21) from manually assigned unambiguous NOEs and dihedral angle constraints obtained from both 3JNH{alpha} coupling constants and the GRIDSEARCH module of DYANA. Zinc-ligating atoms were identified by examination of the initial structures calculated in the absence of zinc. Final calculations were then carried out using ARIA 1.2 (22) implemented in CNS 1.1 (23), using the standard protocols provided, with the zinc geometry and the experimentally determined tautomeric state of the histidine side chains fixed (24). Final assignments made by ARIA 1.2 were checked manually and corrected where necessary. In the final set of calculations, the 100 lowest energy structures were refined in a 9-Å shell of water using standard ARIA 1.2 water refinement modules (minimization and dynamics steps, for details see Refs 25 and 26). The 20 conformers with the lowest value of total energy were analyzed using MOLMOL (27) and PROCHECK_NMR (28).

HADDOCK Docking—F5 was docked to the DNA using the program HADDOCK 1.3 (12-14). The starting structures for the docking were a B-form model of the double helix DNA fragment (5'-ACCGAAAGTTCAC) constructed with the Nucleic Acid Builder package (29) and the 20 lowest energy structures of F5. Based on NMR titration data, disordered sequences consisting of the ten N-terminal and seven C-terminal residues of F5 (and the Zn atom) were excluded from the calculation. Active and passive residues for both the protein and the DNA were chosen based on experimental data and solvent accessibility (>50%) determined by the program MOL-MOL. A 2-Å distance was used to define the ambiguous interaction restraints (AIRs). For the DNA fragment, bases 6-9 and 18-20 located within the consensus sequence were selected as "active" based on the chemical shift changes observed upon complex formation. AIRs were defined solely from the unique base atoms of bases 8, 9, 19, and 20 to suitable atoms (i.e. from H toNorOand vice versa) of all active and passive residues of F5, whereas for bases 6, 7, and 18 the whole base was selected because of the lower specificity of these nucleotides (30). The four flanking nucleotides (bases 5, 10, 17, and 21) were chosen as "passive." For F5 AIRs were defined between all atoms of all active residues (Ile-857, Asn-860, Tyr-861, Ser-863, Arg-865, and Ser-868) and all atoms of all active and passive DNA bases. Passive residues of the protein were defined as the solvent-accessible surface neighbors of active residues (Ser-854 and Ala-862). A total of 18 AIRs resulted from all definitions described above and were used as input in HADDOCK. Additional restraints to maintain base planarity and Watson Crick bonds were introduced for the DNA. During the rigid body energy minimization, 1000 structures were calculated, and the 200 best solutions based on the intermolecular energy were used for the semi-flexible, simulated annealing followed by an explicit water refinement. The solutions were clustered using a cutoff of 3.5-Å r.m.s.d. based on the pair wise backbone r.m.s.d. matrix. The semi-flexible annealing and the water refinement steps of HADDOCK were re-run with the best five structures of the lowest energy clusters (cutoffs 0.9 Å) (30). The final 120 structures were clustered as described above, resulting in a single low energy cluster of 23 structures. The best 10 structures (r.m.s.d. 0.7 Å over backbone atoms) of this cluster were analyzed using standard HADDOCK protocols and were used to represent a model of the complex.


Figure 1
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  		FIGURE 1.
Schematics of MyT1 and DNA. A, schematic representation of mouse MyT1, showing the constructs used in this work. Gray boxes indicate zinc fingers. B, sequence alignment of ZnF domains in mMyT1. Zinc-ligating residues are marked by asterisks, and regions of very high sequence similarity are indicated by gray shading, DNA-binding residues are boxed, and point mutants are indicated by black arrows. Numbers refer to the full-length proteins. C, sequence of the β-RARE oligonucleotide used in this work. Boxed bases are part of the consensus sequence.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The DNA-binding Properties of MyT1 ZnF Combinations—To understand the DNA-binding properties of MyT1 ZnFs, we first expressed and purified polypeptides corresponding to different combinations of the MyT1 ZnFs (Fig. 1, A and B) and tested their ability to bind a 13-bp oligonucleotide containing the β-RARE sequence (5'-ACCGAAAGTTCAC, Fig. 1C)by SPR. Both two-dimensional NMR and circular dichroism melting data confirmed that this oligonucleotide is a well ordered B-form double helix in the SPR buffer (data not shown). The B-form nature of the oligonucleotide was confirmed by analyzing intra- and internucleotide NOE intensities (31).

We first measured the affinity of individual ZnFs for the β-RARE DNA. As shown in Fig. 2A, significant differences in the binding properties were observed between fingers. The data for finger 5 (F5) fitted well to a 1:1 binding isotherm, yielding a KD of 200 µM (see Fig. 2G). Reduction of the salt concentration from 150 to 50 mM increased the binding affinity to 11 µM (Fig. 2H), indicating a significant electrostatic component to the F5-DNA interaction. In comparison the binding of F7 was slightly weaker (KD = 51 µM at 50 mM NaCl) and F3, F4, and F6 bound so weakly that an affinity constant could not be reliably measured. As an alternative (albeit semiquantitative) means of gauging the relative affinities of each ZnF for DNA, we compared the SPR response obtained following the injection of equal amounts of protein onto identically prepared chips (e.g. see inset in Fig. 2A for data on F5 and F7). This approach gives a reliable indication of the relative propensities for binding, provided the protein concentration used is of the order of the dissociation constants (as it was in these experiments). As shown in Fig. 2A, the different ZnFs clearly have different DNA-binding properties in isolation. Overall, F2 and F5 appeared to bind with similar affinities, whereas F3, F4, and F6 bound relatively poorly. F7 exhibits ~50% of F5 binding using this approach (Fig. 2A and inset), in good agreement with the affinities determined above. Interestingly, the affinities of double finger constructs displayed less variation (Fig. 2B).

ITC data revealed that F5 binds to β-RARE with a 1:1 stoichiometry and an affinity of 27 µM (Fig. 2C). Note that these data were recorded under conditions where the so-called "c-value," the product of the association constant and the receptor (in this case the protein) concentration, was ~0.3. ITC data are typically recorded with c in the range 10-300, under which conditions the data are sigmoidal in shape. However, it has been rigorously established that accurate KD values can still be determined for weak interactions where c values are as low as 0.01 or less (32). The binding constant obtained for the F5-DNA interaction agrees well with that obtained by SPR (11 versus 27 µM, see above).

A double finger construct containing both F4 and F5 (F45) also binds in a 1:1 fashion, but two orders of magnitude more tightly (KD = 0.27 µM, Fig. 2D). Thus, binding of the β-RARE probe is dominated by a subset of the MyT1 ZnFs (F2 and F5, and probably also F7), but the other fingers can make a significant contribution to binding at a GAAAGTT site.

Analysis of ZnF-DNA Interactions—We next sought to examine the specificity of DNA binding by MyT1-type ZnFs in more detail. Preliminary NMR data (not shown) indicated that ZnFs 2 and 4, while folded, existed in solution as multiple species that were in slow exchange on the chemical shift timescale. Additionally, SPR binding curves for the F2-DNA interaction could not be fitted by any available kinetic model. Both of these findings are consistent with oligomerization of the proteins; a conclusion that was subsequently confirmed by multiangle laser light scattering (not shown). We therefore focused on the interaction of F5 with DNA.


Figure 2
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  		FIGURE 2.
DNA-binding properties of MyT1 ZnFs. A, binding of different MyT1 ZnFs relative to F5, as calculated from the SPR signal 2 min after injection of protein onto a β-RARE derivatized sensor chip (n = 3-4). Inset, binding curves for F5 and F7, illustrating the data used to calculate relative binding propensities. B, binding of double finger constructs evaluated as described in A (n = 3-4). C, ITC data for the F5-β-RARE interaction. Raw and integrated data are shown, together with the fit to a 1:1 binding model. The fit shown represents a portion of the theoretical curve for a one-site binding event with the given KD. D, ITC data for the F45-β-RARE interaction. E and F, SPR data showing the ability of mutant oligonucleotides to compete with chip-bound β-RARE DNA for the binding of either F5 (E) or F45 (F). For F5, the A7C, G8T, T9G, and T10G mutants compete significantly (p < 0.01) less well than β-RARE. In the case of F45, G4T, A5C, and A6C are also significantly poorer competitors. G, SPR binding curves for the F5-β-RARE interaction (150 mM NaCl). Protein concentrations were 110, 55, 27.5, 13.8, 6.9, 3.4, 1.7, and 0.9 µM. The dashed line represents a fit to the Langmuir 1:1 interaction model. H, SPR binding data for the interaction of F5 with DNA (at 50 mM NaCl), plotted as a binding isotherm by taking the equilibrium response value following the injection of different concentrations of F5. The dashed line shows a fit to a 1:1 binding model, which yielded a KD of 11 µM.

 
To determine the importance of each DNA base for the interaction, SPR competition experiments were performed. β-RARE DNA was immobilized and F5 was injected in the presence of a 2-fold molar excess of a competitor oligonucleotide (Fig. 2E). This series of competitors was based on wild-type β-RARE, with each base mutated in turn (A to C, G to T, and vice versa). Under these conditions, wild-type β-RARE reduced the SPR signal by ~80% and all other competition experiments were compared with this value. The mutants A7C, G8T, T9G, and T10G were significantly less effective competitors for F5 binding than the other oligonucleotides (p < 0.01), indicating that the central AGTT sequence is likely to represent the target site for F5. The same experiment was repeated for the double finger construct F45 (Fig. 2F), whereupon three additional positions (GAA) upstream of the central AGTT motif were demonstrated to contribute to F45 binding. Notably, the changes in relative competition at the AGTT motif were significantly larger for F45 compared with F5 because of the different conditions used (which were restricted by the amounts of protein and DNA available and the binding affinities for the different interactions).

Structure of F5—Next, we used standard 1H- and 15N-separated NMR experiments to determine the solution conformation of F5. Initial structures calculated in DYANA (21) using unambiguous restraint data indicated that Cys-846, Cys-851, His-864, and Cys-870 were the zinc ligands. The structure was then refined in ARIA (22, 33), with the inclusion of restraints to define the zinc geometry (34). The 20 lowest energy structures from a total of 700 were used to represent the structure of F5 (Fig. 3A, PDB code 2JYD). These structures display good covalent geometry and no restraint violations >0.5Å or 5 ° (Table 1). The structure is well defined, with an r.m.s.d. for backbone atoms of 0.41 Å (residues 845-874), but lacks any elements of repeating secondary structure (i.e. {alpha}-helices and β-sheets). Rather, it comprises several irregular loops with a central zinc atom (Fig. 3A). The four zinc-ligating residues, together with Thr-848, Gly-855, Gly-859, and Gly-869, make up a small hydrophobic core. Fig. 3B shows an electrostatic surface representation of F5, revealing a distinct groove on one side and a mixture of positive and negative potential on the opposing surface. A small electrostatic network also appears to exist on one surface of F5, comprising Lys-845, Asp-852, and Arg-872 (Fig. 3B). Mutation of the surface exposed Arg-872 to alanine disrupts the structure of F5, as determined by one-dimensional NMR spectroscopy (data not shown), consistent with a role for these electrostatic interactions in stabilizing F5.


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  		TABLE 1
Structure statistics of F5 and F5·DNA complex

 


Figure 3
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  		FIGURE 3.
Solution structure of F5. A, stereo view of the backbone of the 20 lowest energy structures of F5 (residues 845-874). Residues 837-844 and 875-880 are disordered and are not shown. Heavy side-chain atoms of the zinc ligating residues (Cys-846, Cys-851, His-864, and Cys-870) are shown in green, and zinc atoms are colored yellow. The dimensions of the domain are 21 Å (top to bottom) by 22 Å at the widest point. B, electrostatic surface representation of F5. The structure on the left has been rotated by 45° clockwise about the vertical axis when looking from above relative to A. Important residues have been labeled. (The coordinates for the structure were deposited to the Protein Data Bank with the accession code 2JYD.)

 
Mapping the MyT1-F5·DNA Interaction by NMR Spectroscopy—To determine how MyT1-type ZnFs recognize DNA, we analyzed chemical shift perturbations following a titration of 15N-F5 with β-RARE. Fig. 4A shows a section of the 15N-HSQC spectrum of F5 during the titration. The selective shift of a subset of resonances, together with the fact that no changes were observed in the spectrum following the addition of more than one molar equivalent of DNA (data not shown), indicates a specific interaction between F5 and DNA. The interaction is an intermediate to fast exchange on the chemical shift timescale, as expected for the formation of a complex with a Kd >10 µM. A summary of chemical shift changes is shown in Fig. 4B, and in Fig. 4C residues that both exhibit significant chemical shift changes and have a solvent accessibility >50% (active residues, see below) are mapped onto a space-filling representation of F5. These residues lie mainly on one face of the structure, suggesting that this surface represents the DNA-binding surface of F5. The surface, which is somewhat concave, comprises a mixture of polar and hydrophobic side chains and bears an overall positive charge (Fig. 3B, left, and Fig. 4C).

15N-HSQC titrations were also carried out using double finger constructs F45 and F56 (data not shown). Chemical shift changes for the F56 construct were dominated by changes in F5, whereas in the case of F45 chemical shift changes were observed for signals in both ZnFs. Unfortunately, intermediate chemical exchange combined with a tendency for the double finger constructs to degrade, precluded the detailed analysis of their interaction with DNA.

Analysis of NOESY spectra of the F5·DNA complex also allowed chemical shift changes to be evaluated for protons in the DNA (Fig. 4, D and E). Overall, the largest chemical shift changes were observed for the central AGT portion of the duplex, consistent with the SPR competition experiments.

A Model of the F5·DNA Complex—Although the F5·DNA complex was ~99% populated under the conditions of our NMR experiments ([F5] = [β-RARE] = 1mM), no intermolecular NOEs could be unambiguously identified from either double half-filtered or a combination of two-dimensional [F1,F2] and two-dimensional [F1] 13C,15N-filtered NOESY spectra (35, 36). Notably, resonances from a number of DNA base protons (e.g. GUA8 and CYT19) as well as F5 side-chain protons (including those of Tyr-861) that are likely to form the protein·DNA interface could not be located in spectra of the complex. These signals were most likely experiencing intermediate exchange.

Therefore, to gain insight into the likely DNA-binding mode of F5, we used our chemical shift perturbation and mutagenesis data to create a model of the F5·DNA complex using the data-driven docking program HADDOCK (12-14). HADDOCK comprises a series of Python scripts that run on top of the structure determination programs ARIA (22) and CNS (23); it is designed to use biochemical and/or biophysical interaction data to generate AIRs and to then carry out docking calculations to generate models of a complex that satisfy the experimental data (37-39).

In the current case, active protein and DNA residues were defined as those that both underwent significant changes in chemical shift and were >50% surface exposed (Fig. 4). Six AIRs were defined from active protein residues (Ile-857, Asn-860, Tyr-861, Ser-863, Arg-865, and Ser-868) to 11 active and passive DNA bases (bases 5-10 and 17-21; note that the AAGTT-containing DNA strand is numbered 1-13, and the complementary strand is numbered 14-26). Similarly, 12 AIRs were defined from 7 active DNA bases, on the basis of the DNA specificity data (Fig. 4, D and E).

Following a two-stage docking, simulated annealing, and water refinement protocol (30), the ten lowest energy structures were selected to represent a model of the complex structure (Fig. 5A, PDB code 2JX1). These structures overlay with an r.m.s.d. of 0.7 Å over all backbone atoms of the complex. No restraint violations >0.5 Å were observed in the final structures (Table 1). The DNA-binding surface of F5 comprises mainly residues in the long irregular loop between the two pairs of zinc ligands (Fig. 5, B and C); this loop inserts into the major groove and makes contacts over mainly four base pairs: the AAGT of the consensus sequence. Ten intermolecular hydrogen bonds and 14 hydrophobic contacts are observed in >50% of the structures (Table 2), and the base-specific interactions can be seen in Fig. 5C. The side chains of both Tyr-861 and Ser-868 play a dominant role in DNA recognition: the former makes two hydrogen bonds with THY9 and CYT19, as well as hydrophobic contacts with GUA8 and ADE18, whereas the latter forms hydrogen bonds with GUA8, CYT19, and THY20, as well as van der Waals' contacts to bases ADE7, GUA8, and CYT19. Other hydrogen bonds to base protons are formed by the backbone O of Leu-867, whereas Arg-865 and Ser-866 form hydrogen bonds with atoms in the phosphate backbone. Notably, the chemical shift of the carbonyl carbon of Leu-867 (180.2 ppm) is significantly high compared with other residues, which agrees well with the observation that the carbonyl oxygen of Leu-867 forms a hydrogen bond with base protons of both ADE6 and ADE7.


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  		TABLE 2
Intermolecular interactions between F5 and DNA

The specific interactions in are in bold.

 
The conformation of the DNA after the docking process was analyzed using the program 3DNA (40). Overall, the DNA remained double-stranded throughout the calculation, although some deviations from canonical base-pairing geometry were observed in the protein-binding region (ADE6-THY10, Fig. 6). The width of the major groove of F5-bound DNA is at a maximum between ADE7-GUA8 and GUA8-THY9 indicating the weak intercalation of residues Ser-868 and Tyr-861 (Fig. 5B), respectively.

In an effort to validate our model, we constructed point mutants of F5, targeting residues Tyr-861 and Ser-868, which form the majority of specific hydrogen bonds with the DNA (Table 2) as well as Thr-858, Asn-860, Arg-865, and Arg-872. One-dimensional 1H NMR spectra of alanine mutants T858A, Y861A, S863A, R865A, S868A, and R872A revealed that they were unfolded; however, the more conservative mutations Y861F, S868V, N860A, and S866D, which all target the proposed DNA-binding face of F5, folded correctly according to one-dimensional 1H NMR spectra and all resulted in significant decreases in DNA binding (Fig. 7).

Finally, we noted that, although the DNA binding ability of F4 and F5 is significantly different (Fig. 2A), the amino acid sequence of the two domains is conserved at the DNA binding interface (Fig. 1). In an attempt to explore the reason leading to this difference, Lys-845 was mutated to alanine. This residue is one of the few in the F5 structure that is not present in F4 (the corresponding residue is Thr-801). The mutation resulted in a decrease of DNA binding compared with F5 (Fig. 7) suggesting that the electrostatic network between Lys-845, Asp-852, and Arg-872 plays a role in stabilizing the protein·DNA complex.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The DNA Binding Mode of MyT1 Zinc Fingers—In this study, we have sought to understand the molecular basis for DNA recognition by MyT1-type ZnFs. Our data define the footprint made on the DNA by a single MyT1 ZnF and suggest the molecular basis for the DNA-binding properties of these domains.

The model that we have constructed from a combination of NMR and SPR binding data indicates that MyT1 ZnFs form small, compact structures that can sit entirely within the major groove of DNA. The side chains of a small subset of residues (primarily Tyr-861 and Ser-868) make specific contacts with DNA bases in the 4-bp AAGT portion of the MyT1 consensus sequence, and these interactions most likely contribute to specificity as well as binding affinity. The weak intercalation of Tyr-861, through hydrogen bonding of the terminal OH group and van der Waals' interactions of the aromatic ring, probably plays a significant role in the recognition of the DNA (Fig. 5 and Table 2). Tyrosine intercalation during DNA binding has been observed with other non-ZnF proteins (41).

Our model reveals that there is good shape complementarity between MyT1 ZnFs and the DNA major groove (Fig. 5). The positioning of the domain in the major groove corroborates published methylation interference data obtained on the homologous rat NZF1 protein, which also indicated an interaction with the major groove (10, 42).


Figure 4
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  		FIGURE 4.
NMR analysis of the F5-DNA interaction. A, portion of the 15N-HSQC of 15N-F5 during titration with β-RARE DNA. Black: 0 eq. DNA, blue: 0.25 eq. DNA, green: 0.5 eq. DNA, and orange: 1 eq. DNA. The initial concentration of F5 was 1.3 mM. B, upper panel: weighted chemical shift changes for F5 backbone amides (HN, N) calculated from n = 3 HSQC titrations; middle and lower panels: weighted chemical shift changes for C{alpha} and C'. Filled bars represent significant (average plus one standard deviation) changes; no bar indicates the absence of the corresponding signals. The dotted line indicates the average chemical shift change ± one standard deviation. C, space filling representations of F5 with likely binding residues ({Delta}{delta} > one standard deviation and solvent accessibility >50%) colored in red. Orientation is the same as the left-hand panel of Fig. 3B. D, analysis of chemical shift changes for DNA protons during the titration with F5. The total height of each bar represents the total number of non-equivalent proton signals per base that could be unambiguously assigned in the spectra of both DNA alone and the complex with F5. Filled bars show the number of protons signals for which chemical shift changes were significantly higher than the average or where disappearance of the corresponding signal upon complex formation was evident from the spectra. Signals originating from base protons are colored in black and sugar protons in gray. E, space filling representation of the DNA with active bases (see text for definition) colored in red.

 
During HADDOCK calculations, we often observed an alternative cluster of structures in which the same protein residues made interactions with the DNA (predominantly Tyr-861, Ser-866, and Ser-868), but where the orientation of F5 in the major groove differed by 180°. In these complexes, the general fit of the domain into the major groove was good, but a statistical analysis of calculated CNS energies (for details see Ref. 23) revealed a significantly higher (p < 0.01) energy compared with our model ({Delta}~ 70 kcal/mol). In addition, distance violations were observed in all of these structures (>0.5 Å), arguing against this alternative structure being correct.

Although many zinc fingers recognize the major groove of DNA, the mechanism through which MyT1 achieves this end, insertion of the entire domain in the groove, contrasts sharply with the classical, GATA-type, and steroid hormone receptor ZnFs. These latter domains all utilize an {alpha}-helix to position DNA-contacting residues appropriately and in fact many other DNA-recognition domains, including homeodomains and other helix-loop-helix proteins, take advantage of the size match between the {alpha}-helix and the DNA major groove. MyT1-type ZnFs are found in far fewer proteins than classical ZnFs, and in this regard are more similar to folds such as GATA-type, TAZ, and RanBP-type zinc finger domains, which are also comparatively rare.

Weak versus Nonspecific Interactions—Our data show that the binding of either single or double MyT1 zinc finger constructs to DNA is significantly weaker than that typically observed for sequence-specific DNA-binding proteins (~1-10 nM). However, it is important to emphasize that, despite its low affinity, the MyT1-DNA interaction is sequence specific: our SPR competition experiments showed that even single base mutations significantly reduced the binding of F5 (or F45) to β-RARE. Interestingly, a recent analysis of genome-wide ChIP-on-chip data from yeast (43) suggested that weak protein-DNA interactions are extremely abundant and are likely to be functional.

More generally, it is often tempting to use the terms "weak" and "nonspecific" interchangeably when discussing bimolecular interactions, but it is important to point out that these descriptors are definitely not equivalent. The interaction between MyT1 and DNA is relatively weak, but a wide variety of experimental data demonstrates that it is highly specific. For example, the F5·DNA NMR titration was saturable, point mutations to the DNA sequence disrupted binding, and the SPR response reached equilibrium following each of the protein injections (Fig. 2G).


Figure 5
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  		FIGURE 5.
Model of the F5·DNA complex structure. A, the 10 lowest energy structures from HADDOCK are shown (F5 as a backbone trace). B, surface representation of the DNA and ribbon diagram of F5 depicting the insertion of the protein into the DNA major groove. The side chains of Tyr-861, Leu-867, and Ser-868, which make base-specific contacts, are shown in red. C, representation of the lowest energy structure of the F5·DNA complex (same orientation as in A). Heavy atoms of side chains of Zn ligating residues are colored green and the zinc atom is yellow. To show the binding of F5 in more detail, a larger image of the interacting region is shown. DNA bases involved in specific interactions are colored blue and protein residues red; interactions are indicated as black dashed lines; spheres represent O and H atoms involved in DNA-specific interactions.

 
A related point is that it is often presumed that weak interactions are unlikely to be physiologically relevant because the relevant species will not be present in the cell at the required concentrations. However, there is an abundance of data in the literature that contradicts this assumption. For example, protein-protein interactions, such as those involving either NZF zinc-finger domains and ubiquitin (44), the transcription factors GATA-1 and FOG (45), or modified histone tails and their recognition domains (e.g. plant homeo domain (PHD) and chromo- and bromo-domains (46-48)), frequently have dissociation constants in the 1-10 µM range.

Given that interactions described above are known to be important biologically, the simplest conclusion that can be drawn from these data is that binding affinities measured in vitro do not fully reflect the situation inside a cellular compartment, where for example, local concentrations might be drastically altered by compartmentalization, subcellular targeting processes, or the formation of large multiprotein complexes. Thus, in vitro binding affinities are a useful relative measure (when comparing different domains or a panel of mutants, for example), but the relevance of their absolute values is difficult to gauge.

Multi-ZnF Proteins and DNA Recognition—The current study has improved our understanding of how the multiple zinc fingers of MyT1 combine to recognize DNA. SPR analysis shows that, although neither F4 nor F6 bind well to DNA, they can each combine with F5 to increase the affinity of MyT1 for DNA. Our data indicate that the double finger construct F45 is likely to make base-specific contacts across the full length of a GAAAGTT site (Fig. 2F), indicating that a two-finger unit is both necessary and sufficient to obtain full recognition of the β-RARE site. Given the high similarity between the sequences of F4 and F5 and the partially palindromic nature of the DNA site, it is likely that F4 of the double finger construct contacts the GAA sequence in an orientation that differs by 180° from that observed for F5 binding to AGTT. This idea is consistent with our model of the F5·DNA complex, wherein the N terminus of F5 points toward the 5'-end of the β-RARE site (Fig. 5), as well as with the measured 1:1 binding stoichiometry of F45 (Fig. 2D) and the SPR competition experiments (Fig. 2F).


Figure 6
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  		FIGURE 6.
Averaged helical parameters for β-RARE DNA bound to MyT1 F5 (solid lines) as calculated with 3DNA. Twist (squares), roll (dots), and width of the major groove (triangles) are shown. The corresponding values for the initial (computer generated) B-form DNA duplex are indicated for comparison (dotted lines).

 


Figure 7
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  		FIGURE 7.
Relative binding of different MyT1 ZnFs mutants as calculated from SPR resonance units 2 min after injection of protein onto a β-RARE derivatized sensor chip (n = 3-4).

 
The DNA recognition site identified for MyT1 (4) in the human PLP promoter contains a single copy of an AGTTT sequence and is preceded by three purines (GGA). Earlier studies also suggested that purines upstream of the AGTT site are crucial for DNA binding (10). This site is located 256 bases upstream of the coding region of the PLP1 and is 100% conserved in the chimp. Despite the fact that mouse and rat MyT1 share a high homology with the human protein (5), particularly in the DNA-binding region identified in this study, a corresponding target site could not be located at the same position in these genomes. However, a very similar site was found 777 bases upstream of the PLP1 start site, comprising an AGTTT site preceded by three purines (AAG). It is quite possible that this represents the target site for MyT1.

Yee and Yu (11) have found that NZF-3, the third member of the MyT1 family, binds substantially better to a DNA probe containing two repeated AAAGTTT motifs. However, the reported gel shift experiments were carried out using glutathione S-transferase fusions of MyT1 zinc finger domains, and we have observed in other contexts that the propensity of glutathione S-transferase to dimerize can lead to such results.7

Given that there are no tandem AAAGTT sites within the proximal promoter of the human or rodent PLP1 gene, the question arises as to why MyT1 carries three double-ZnF domains (F45, F67, and the distant F23). It is possible that distinct two-ZnF clusters might affect chromatin structure by binding simultaneously to highly separated DNA sites. This possible activity is interesting in light of the recent finding that MyT1 can recruit Sin3B (49) and could indicate a mechanism in which the binding of MyT1 to distant sites and the co-opting of Sin3B results in the reorganization of local chromatin structure. Mechanisms of this type have long been suggested for many transcriptional activators, including multi-ZnF proteins that can contain up to nearly 40 classic zinc fingers, but we still have very little direct evidence of such activities.

In conclusion, we have used a range of biophysical methods to delineate the mechanism of DNA recognition by the unusual MyT1 class of zinc fingers. Our results indicate that MyT1 ZnFs recognize 3-4 bp of double-stranded DNA in a sequence-specific manner by inserting the whole domain into the major groove of the DNA. These data further our understanding of protein-DNA recognition.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2JX1 and 2JYD) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by a National Health and Medical Research Council (NHMRC) program grant (to J. P. M. and J. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Supported by a fellowship from the Austrian Science Fund (Grant J2474). Back

3 Supported by a PHD (plant homeodomain) scholarship from Dr. Geoff Duggin. Back

4 A Viertel Medical Research Fellow. Back

5 An NHMRC Senior Research Fellow. To whom correspondence should be addressed. Tel.: 61-2-9351-3906; Fax: 61-2-9351-4726; E-mail: j.mackay{at}mmb.usyd.edu.au.

6 The abbreviations used are: ZnF, zinc finger; MyT1, myelin transcription factor 1; PLP, proteolipid protein; NZF1, neuronal zinc finger 1; β-RARE, β-retinoic acid receptor gene; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; F5, Finger 5; MES, 4-morpholineethanesulfonic acid; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; AIR, ambiguous interaction restraint; r.m.s.d., root mean square deviation; ChIP, chromatin immunoprecipitation. Back

7 F. Loughlin and J. P. Mackay, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Alexandre Bonvin and Marc van Dijk for assistance with the docking calculations and Lynn Hudson for the original mMyT1 construct.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. A. Lowry, R. Gamsjaeger, S. Y. Thong, W. Hung, A. H. Kwan, G. Broitman-Maduro, J. M. Matthews, M. Maduro, and J. P. Mackay
Structural Analysis of MED-1 Reveals Unexpected Diversity in the Mechanism of DNA Recognition by GATA-type Zinc Finger Domains
J. Biol. Chem., February 27, 2009; 284(9): 5827 - 5835.
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