Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12123–12128, October 1998

Crystal structure of the BTB domain from PLZF

K. FARID AHMAD,CHRISTIAN K. ENGEL, AND GILBERT G. PRIVE´*

Division of Molecular and Structural Biology, Ontario Cancer Institute, and the Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9

Communicated by David Eisenberg, University of California, Los Angeles, CA, August 19, 1998 (received for review July 9, 1998)

ABSTRACT The BTB domain (also known as the POZ (7), we estimate that 5–10% of zinc finger also contain domain) is an evolutionarily conserved –protein in- BTB domains. The domain is known to form homomeric and teraction motif found at the N terminus of 5–10% of C2H2-type heteromeric associations with other BTB domains (3), and zinc-finger transcription factors, as well as in some actin- given the large number of BTB domain proteins in the associated proteins bearing the kelch motif. Many BTB pro- genomes of higher eukaryotes, an attractive possibility is that teins are transcriptional regulators that mediate gene expres- the domain generates a combinatorial diversity of complexes sion through the control of chromatin conformation. In the within a family of BTB domain proteins. human promyelocytic leukemia zinc finger (PLZF) protein, The organization of the human promyelocytic leukemia zinc the BTB domain has transcriptional repression activity, di- finger (PLZF) protein is typical of BTB domain proteins: the rects the protein to a nuclear punctate pattern, and interacts 120 BTB domain is found at the N terminus of the with components of the deacetylase complex. The protein, followed by a central region of several hundred amino association of the PLZF BTB domain with the histone deacety- acids, and ending with a series of C2H2 Kruppel-type zinc lase complex provides a mechanism of linking the transcrip- fingers. The PLZF protein is a potent transcriptional repressor tion factor with enzymatic activities that regulate chromatin implicated in embryonic development and in hematopoesis (8, conformation. The crystal structure of the BTB domain of 9). It occurs as a fusion protein with the retinoic acid PLZF was determined at 1.9 Å resolution and reveals a tightly ␣ (RAR␣) in the rare t(11;17) form of acute promyelocytic intertwined dimer with an extensive hydrophobic interface. leukemia (8). In this acute promyelocytic leukemia, the BTB Approximately one-quarter of the monomer surface area is domain from PLZF is responsible for the dominant negative involved in the dimer intermolecular contact. These features properties of the PLZF-RAR␣ fusion against wild-type are typical of obligate homodimers, and we expect the full- RAR␣-retinoic acid X receptor (RXR␣) function (10). Cells length PLZF protein to exist as a branched transcription from the more common t(15;17) PML-RAR␣ acute promy- factor with two C-terminal DNA-binding regions. A surface- elocytic leukemia can be induced to differentiate with treat- exposed groove lined with conserved amino acids is formed at ment with retinoic acid (RA), but PLZF-RAR␣ cells are the dimer interface, suggestive of a peptide-binding site. This responsive to RA only if potentiated with inhibitors of the groove may represent the site of interaction of the PLZF BTB histone deacetylase complex (11–14). Because the BTB do- domain with nuclear corepressors or other nuclear proteins. main from PLZF can associate with mSin3A, histone deacety- lase 1 (14), SMRT (silencing mediator for retinoid and thyroid The BTB domain (Broad-Complex, Tramtrack, and Bric` a hormone receptors) (13), and N-CoR ( core- brac) (1, 2), also known as POZ (poxvirus and zinc finger) (3), pressor) (12), the PLZF–RAR␣ fusion protein has been is an evolutionarily conserved protein–protein interaction proposed to repress transcription at RA-sensitive sites through domain often found in developmentally regulated transcrip- the BTB-mediated recruitment of the histone deacetylase tion factors. The domain is strongly implicated in the regula- complex (11–14). tion of gene expression through the local control of chromatin Because the domain is widely represented in eukaryotic conformation (4). The domain was first identified in a set of genomes, features common to BTB proteins provide crucial Drosophila and poxvirus genes (5), and examples of BTB hints as to the function of the domain. Punctate nuclear domain genes have since been found in organisms ranging staining patterns are commonly seen in zinc-finger BTB pro- from yeast to man. teins (4), and for ZID, PLZF, and BCL6 (or LAZ3), this has A search of the current publicly available sequence data- been shown to require an intact BTB domain (3, 10, 15). Many bases reveals 56 distinct human BTB entries, of which 22 BTB domain proteins, including Tramtrack, PLZF, and BCL6, correspond to named, full-length genes, whereas the remaining are transcriptional repressors, although some, such as GAGA entries are known only as tentative human consensus (THC) factor, can counteract repression by chromatin remodeling sequences, or expressed sequence tags (EST), (a tabulation of (16). As with other conserved domains, we expect that the these genes can be found at http:͞͞xtal.oci.utoronto.ca͞prive͞ domain carries out a common underyling molecular role, btbtable.html). Approximately two-thirds of the full-length which can be used for a variety of biological functions. Our human BTB genes also encode C2H2 zinc finger modules, structure of the PLZF BTB domain reveals that the BTB whereas approximately one-half of the remaining entries con- domain is a tightly interwound dimer and identifies sites at the tain the kelch motif (4, 6). None of the known human BTB dimer interface, which may be important for BTB–BTB do- genes contain more than one single copy of the domain, main interactions, as well as surface features in the dimer, making it likely that most, if not all, of the known BTB which may be involved in interactions with other proteins. tentative human consensus (THC) and expressed sequence tag (EST) entries correspond to distinct genes. Based on the Abbreviation: PLZF, promyelocytic leukemia zinc finger; 3D, three- projection that there are 300–700 zinc finger proteins in man dimensional; CHESS, Cornell High Energy Synchrotron Source; RA, retinoic acid; RAR␣, RA receptor ␣; SMRT, silencing mediator for The publication costs of this article were defrayed in part by page charge retinoid and thyroid hormone receptors. Data deposition: The atomic coordinates have been deposited in the payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Data Bank, Biology Department, Brookhaven National Lab- accordance with 18 U.S.C. §1734 solely to indicate this fact. oratory, Upton, NY 11973 (PDB ID code 1BUO). © 1998 by The National Academy of Sciences 0027-8424͞98͞9512123-6$2.00͞0 *To whom reprint requests should be addressed. e-mail: prive@oci. PNAS is available online at www.pnas.org. utoronto.ca.

12123 Downloaded by guest on October 2, 2021 12124 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 95 (1998)

METHODS thionine-substituted protein and the structure was determined by multiwavelength anomalous diffraction phasing (Tables 1 Codons 1–132 from a human PLZF cDNA (gift from J. D. and 2). The structure reveals a tightly intertwined homodimer, Licht, Mount Sinai School of Medicine, New York) were consistent with hydrodynamic studies (22). The dimer can be amplified by PCR and subcloned into the plasmid pET-32(a), described roughly as a prolate ellipsoid with overall dimensions producing an expression vector for an N-terminal thioredoxin of Ϸ60 Å by 26 Å by 32 Å. Because of the flattened shape of domain followed by a 6X His tag sequence, a 40-aa linker, and amino acids 1–132 of PLZF. The selenomethionyl-substituted the molecule, most residues are at or near the surface of the protein. The central scaffolding of the protein is made up of a fusion protein was overexpressed in the Escherichia coli me- ␣ ␤ thionine auxotroph strain B834(DE3). After cell lysis and cluster of -helices flanked by short -sheets at both the top protein purification by nickel chelate affinity chromatography, and bottom of the molecule (Figs. 1 and 2). By using the DALI the peak fractions containing the fusion protein were pooled server (25), a search for proteins with similar three- and dialyzed against 100 mM NaCl, 50 mM Tris⅐HCl (pH 7.5), dimensional (3D) structures revealed no significant entries in 2.5 mM CaCl2, and 0.5 mM Tris [2-carboxyethyl]phosphine the Brookhaven Protein Data Bank (PDB). The N terminus of hydrochloride (TCEP), and trypsin was added to the fusion each chain is associated with the main body of the other chain, ␤ protein in digest buffer at a ratio of 1:1000 (wt͞wt) to remove generating a two-stranded antiparallel -sheet between strand the sequences N-terminal to the BTB domain. Under these ␤1 of one monomer and strand ␤5Ј of the other. (For ease of conditions, the domain was resistant to trypsin treatment for discussion, positions on the symmetry-related molecule will be 24 hr at room temperature. The PLZF BTB domain was labeled with primes. In addition, equivalent symmetry-related purified from the digest mixture by anion exchange chroma- pairs of interactions, such as ␤1͞␤5Ј and ␤1Ј͞␤5 will not be tography and size exclusion chromatography. The purified explicitly mentioned.) Helix ␣6 and the ␤1͞␤5Ј-sheet, along protein was concentrated to 8 mg͞ml in 300 mM NaCl, 50 mM with the symmetry-related elements, form an extended, con- Tris⅐HCl (pH 7.5), and 1 mM TCEP. The final protein cave surface on the underside of the protein dimer. Both chains fragment consists of amino acids 6–132 of PLZF, as confirmed begin and end at the base of the dimer, but because of the by Edman N-terminal amino acid sequencing and ion spray swapped amino termini, the N terminus of one chain is next to mass spectrometry. Crystals of PLZF(6–132) were grown at the C terminus of the other (Fig. 2). This places the two C 4°C in hanging drops by mixing 2 ␮l of protein with 2 ␮lof termini at opposite ends of the base of the dimer, separated by reservoir buffer (0.15 M CaCl2, 0.1 M Hepes (pH 7.5), and 4% a distance of Ͼ58 Å. In intact PLZF, the remaining C-terminal isopropanol), and equilibrating with 1 ml of reservoir buffer regions of the protein would extend from these points. ϭ ϭ for 48 hr. The space group was I4122 (a 72.63 Å, b 72.63 A crystal contact between the lower sheets of two symmetry ϭ Å, and c 168.98 Å) with one BTB monomer in the related BTB dimers generates a short 4-stranded antiparallel asymmetric unit. Crystals were cryoprotected in reservoir ␤-sheet involving four different peptide chains. This associa- solution enriched in CaCl2 to 0.18 M and MPD to 20.0% before tion is probably not biologically important because in solution flash-freezing to 100 K. A multiwavelength anomalous dif- we have only observed dimers at all protein concentrations. fraction experiment was performed at the Cornell High En- This result is consistent with the findings of Li et al. (22) on a ergy Synchrotron Source (CHESS) on beamline F-2, using an PLZF molecule having only slightly different termini than our Area Detector Systems Quantum-4 Charge-Coupled Device construct. (CCD) detector (17). Data processing and reduction were Because the BTB domain self-associates into a dimer and done with MOSFLM and SCALA (18). The positions of 4 of the forms complexes with other proteins, we discuss three struc- 6 expected selenium atoms were determined by both Patterson tural classes of residues in the molecule: those buried within a and Direct methods by using SHELXS-97. The selenium posi- monomer, those buried at the dimer interface, and residues tions were refined with SHARP (19), and three more selenium sites were found by examining residual log-likelihood gradient which are exposed on the surface. Monomer Core. maps after initial refinement runs. Met-58 displays multiple As expected, most of the strongly conserved side chain conformations, accounting for the extra selenium residues are located within the core of the monomer (Fig. 1). site. The final multiwavelength anomalous diffraction phase Especially conserved are His-48, Leu-52, Ser-56, and Tyr-88, set had an overall figure of merit of 0.72 for the 17,622 and these buried residues are all in contact with each other and reflections in the resolution range from 20 to 1.9 Å. The not involved in either the dimer interface, nor exposed to the resulting electron density maps were solvent flattened with DM protein surface. (18), producing maps with clear, connected density for the Dimer Interface. The residues at the intermolecular inter- main chain atoms 6–126 except residue 66 and for 113 of the face determine the specificity and stability of the dimer and are remaining 120 side chains. An atomic model was built into the of particular interest because of the possibility of the formation density with the program O (20) and refined with REFMAC (21). of heterodimers between BTB domain proteins. Interface The final model contains all atoms for residues 6–126 of PLZF. amino acids are distributed throughout the entire length of the The last six residues (amino acids 127–132) are disordered in protein (Figs. 1 and 3). Of the 8,153 Å2 of solvent accessible the crystal and are not visible in the electron density map. surface area in the monomer, 1,999 Å2 are involved in the dimer surface contact. Because of the interchanged ␤1 seg- ment of the monomers, the structure is suggestive of a 3D RESULTS domain-swapped dimer (27). Using the terminology used for General Description of the Structure. We have expressed 3D domain-swapped proteins, the intersubunit contacts can be and crystallized the BTB domain from PLZF as a selenome- grouped into two classes (27): a pair of domain-swapped

Table 1. Diffraction data

Wavelength, Å Resolution, Å Reflections, total͞unique Completeness, % Rsym *, % ͗I͗͘͞␴(I)͘† ␭1 ϭ 0.9793 20.0–1.90 204897͞18253 99.3 (96.7) 4.4 (24.8) 11.2 (3.0) ␭2 ϭ 0.9790 20.0–1.90 189702͞18254 99.4 (97.3) 4.1 (26.9) 12.0 (2.9) ␭3 ϭ 0.9638 20.0–1.90 156050͞17398 94.7 (92.2) 4.3 (25.7) 11.4 (2.8) Parentheses denote statistics in the 2.0–1.9 Å resolution shell. *Rsym ϭ 100 ϫ (¥h¥i(Ih,i Ϫ Ih))͞¥h¥iIh,i for the intensity I of i observations of reflection h. Ih is the mean intensity of the reflection. †͗I͗͘͞␴(I)͘ϭmean intensity͞mean standard deviation. Downloaded by guest on October 2, 2021 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12125

Table 2. Data used in the refinement and refinement statistics strand ␤1. The sequence conservation is preserved in the ␤5 ␤ Resolution, Å 20.0–1.9 region of these proteins, even though the lower -sheet cannot exist in these domains. It remains to be seen what the effects Data cutoff F͞␴(F) 0.0 of this shortened N terminus are on the dimer. Number of Reflections 17,266 The central open interface has a total buried surface of 891 Completeness, % 94.0 Å2 and has contributions mainly from ␣1 and ␣2. Interface R value* 21.2 residues His-16, Pro-17, Leu-20, Leu-21, Lys-23, Ala-24, and R † 25.2 free Met-27 are all located on one face of ␣1, but most of the Number of water molecules 130 contacts are located in the first half of the helix because the rmsd, bond distances, Å 0.021 second half is mostly associated with the main body of the rmsd, angle distances, Å 0.042 individual monomer fold. Two short helices, ␣2 and ␣3, form ͗B͘ (Å2) main chain protein atoms 28.9 ␣ ␣ Ј ͗ ͘ 2 a hairpin with helix 2 packed against the lower portion of 1 , B (Å ) all protein atoms 33.4 such that His-16Ј through Leu-20Ј are sandwiched between ͗ ͘ 2 B (Å ) water atoms 44.5 helices ␣1 and ␣2 (Fig. 2). The sequence of the short ␣2 helix *R value ϭ 100 ϫ ¥͉Fobs Ϫ Fcalc͉͞¥͉Fobs͉, where Fobs and Fcalc are the is one of the most conserved regions in the BTB family, and observed and calculated structure factor magnitudes, respectively. residues from this region make contributions to the monomer † Rfree is the same as the R value but is calculated from 5% of the core, the dimer interface, and the external surface. This region reflection data excluded from the refinement. of the dimer interface is predominately nonpolar in nature, involving mostly contacts between hydrophobic groups. For ‘‘closed interfaces’’ involving ␤1͞␤5Ј͞␣6Ј and ␤1Ј͞␤5͞␣6 and ␥ example, the -CH2 of residue Glu-60 is packed against the a central ‘‘open interface’’ with contributions mainly from ␣1, ␧ ␣ Ј ␣ ␣ Ј -CH2 of Met-27, leaving its charged carboxylate group fully 1 , 2, and 2 . We emphasize that there is no evidence of exposed to the exterior solvent. exchange between monomeric and dimeric forms of the pro- The contacts of the central interface form a closed cavity tein (22), and confirmation that the BTB dimer fold is the with an interior volume of 213 Å3 in the center of the dimer. product of 3D domain-swapping in an ancient precursor will A total of three ordered water molecules have been located in require the knowledge of a monomeric protein with the same this largely apolar cavity, two of which hydrogen bond to each ␤ fold but with the swapped 1 element as part of its main other but make no polar contacts with protein atoms. The rim domain. Although this has yet to be described for this fold, the of this cavity is formed by residues 16, 17, 20, 21, 23, 27, 33, 50, general organization of the dimer is very similar to that of and 54 and sequesters residues Ala-24͞24Ј and Val-51͞51Ј other systems, which have met this criteria for 3D domain- from the external solvent. Such cavities are common in inter- swapping. The probable ‘‘hinge-loop’’ region that would exist subunit contacts (29). The only polar protein atoms in this in a different conformation in the case of a monomeric BTB cavity are the hydroxyl groups of Thr-50 and Thr-50Ј, which fold consists of amino acids 16–19, and these are well ordered hydrogen bond to each other, and the main chain carbonyl and form multiple bridging contacts in the PLZF BTB dimer. oxygens of Pro-17 and 17Ј, which are bridged by the third In the closed interface, one side of the ␤1͞␤5Ј sheet is interior water. Remarkably, the Thr-50 interaction is the only exposed to solvent, and the other side is packed against helix direct polar bond across this central interface, and yet this ␣6Ј. Here, the five main chain H-bonds of the antiparallel amino acid is not conserved in the family of BTB domains (Fig. ␤-sheet contribute to the dimer stability, as well as the burial 1). of the hydrophobic residues Ile-9, Leu-11, Leu-92Ј, and Ala- Potential Ligand-Binding Groove. A grouping of conserved 94Ј that point into the protein interior. It is notable that a few residues at a protein surface is a common feature of protein BTB protein such as Miz-1 (28) and most of the poxvirus functional sites, and next we consider residues that are exposed proteins (5) have a shortened N terminus and thus cannot form to the bulk solvent and have sequence conservation. A cluster

FIG. 1. Sequence alignment of selected BTB domains and the observed secondary structure of the PLZF BTB domain. TTK (Tramtrack) and GAGA factor are Drosophila proteins, and the others are human proteins. The numbering is according to PLZF, and the crystal structure consists of residues 6–126. Black and gray backgrounds are used to indicate identical and͞or conserved residues found in at least 50% of the proteins at a given position. Residues in a 310 helix conformation are indicated by hatching. F indicates amino acids that are classified as buried in the PLZF BTB monomer according to the criteria of Rice and Eisenberg (23). Colored numbers represent the percentage of contribution of the residue to the buried interface surface in the dimer, rounded to the nearest integer (i.e., a value of ‘‘0’’ represents a contribution from 0.1 to 0.49%, values of 1 represent a contribution of 0.5–1.49%, etc.). Residues indicated with red numbers participate in the swapped or closed interface, and amino acids involved with the central open interface are labeled in blue. Residues 16–19 contribute to both and are not classified. Downloaded by guest on October 2, 2021 12126 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 95 (1998)

FIG.2. (A) Ribbon diagram of the PLZF BTB domain dimer. One monomer is colored red and the other blue. The secondary structure elements of one monomer are indicated, and the position of the conserved residue Asp-35 is labeled. In this view, the two halves of the dimer are related by a vertical twofold symmetry axis. This figure was FIG. 4. Sequence conservation in the exposed surface of the BTB generated with the graphical program SETOR (24). (B) Schematic dimer. (A) View in the same orientation as in Fig. 2A.(B) View representation of the topology of the dimer. ␣-Helices are indicated by looking directly down the twofold axis of the dimer. A multiple rectangles, and ␤-sheets by thick arrows. The symmetry related sequence alignment was constructed as follows: residues 6–126 of secondary structure elements are indicated with primes. PLZF were used in a FASTA3 (38) search of the SWALL database of conserved, exposed residues from three different loops is (Nonredundant Protein sequence database including Swissprot, found at the top of the dimer (Fig. 4). The floor of this groove Trembl, and TremblNew) at the EMBL- European Bioinformatics Institute server (http:͞͞www2.ebi.ac.uk͞fasta3). Entries with E score Ͻ0.1 were used for further processing. Identical and closely matching sequences were removed from the set, so that the no two pairs in the final set of 42 sequences had Ͼ88% sequence identity. The set was then aligned, and the sequence variability was calculated as the number of different amino acids present at each residue position. The sequence variability was then displayed on the solvent accessible surface of the dimer by using GRASP (26). In this variability scoring scheme, a fully conserved residue is assigned a value of 1, and the maximum variability is 20. The only exposed, fully conserved residue in the structure is Asp-35, present in the center of the groove.

is made up of residues in the ␣1͞␤2 and ␤3͞␣2 loops (Leu-33, Cys-34, Asp-35, and Arg-49), and the walls of the cleft are formed by the ␣3͞␤4 loop (Phe-63, His-64, Asn-66, Ser-67, and Gln-68). The groove is centered about the twofold axis of the dimer, and the equivalent residues from both chains contribute to the formation of this site. Thus, the groove exists only in the dimer. Asp-35 is adjacent to Arg-49, but the two residues do not form a salt bridge. Instead, the guanidinium group of the arginine forms hydrogen bonds to the main chain carbonyl FIG. 3. View of one monomer displayed as a solvent accessible atoms of Asp-35 and Ser-67 from the same monomer. Asp-35 surface in GRASP (26). The orientation is roughly the same as for the is 37% exposed to solvent, but Arg-49 has only 10% of its blue monomer in Fig. 2A. The surface buried upon dimer formation surface area exposed and is classified as a buried residue by the is indicated in magenta, and residues that contribute at least 2% of the buried surface of the dimer (Fig. 1) are labeled. Residues from the criteria of Rice and Eisenberg (23). The groove is largely closed interface are indicated with arrows. Not shown in the diagram electroneutral as a result of the close proximity of Asp-35 and are the residues from ␤5 and ␣6, which form a groove on the underside Arg-49. From over 100 BTB domains known to date, Asp-35 of the monomer that accommodates ␤1Ј from the adjoining monomer. is absolutely conserved, whereas position 49 is either a lysine The entrance to this groove is lined with Ala-90 and Tyr-113. or an arginine in all but two examples. Downloaded by guest on October 2, 2021 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12127

The conservation data presented in Fig. 4 is based on a binding to putative biological DNA-binding sites is not likely multiple sequence alignment of the 42 highest scoring se- to induce DNA bending or looping unless the two sites are very quences to the PLZF BTB domain and may mask patterns seen widely separated. This also explains the observation that the in more restricted subsets. However, essentially the same result BTB domain can function as an autonomous transrepression was seen when the subset of the 10 most similar human module, and maintains its activities when placed in a variety of sequences was used (data not shown). This does not prove that DNA-binding domain contexts. The presumed flexibility in the other conserved surface features are not found in specific relative positions of the DNA-binding domains also may be subsets of BTB domains, but it does support the existence of important for binding to nucleosomal DNA. a conserved surface common to most, if not all BTB proteins. At the molecular level, the BTB domain has two roles: it can As expected in the constricted space of this groove, there is self-associate to form a dimer, and it can interact with other a well defined and regular array of water molecules in this non-BTB domain proteins. Because the conserved groove in pocket. This is a general feature of binding sites in proteins the BTB domain is at the dimer interface, we predict that (30). The groove is Ϸ8 Å wide and 20 Å long and is of a dimer formation is necessary for the proposed interactions sufficient size to accomodate a 5–6 amino acid peptide. We within this groove. We do not rule out the possibility that that suggest that the binding groove is a potential interaction site the domain contains other protein interaction sites, but these for mSin3A, histone deacetylase 1, SMRT, or N-CoR. It also sites would not have the striking sequence conservation seen is possible that this is a binding site for other, as yet uniden- in the central groove. The PLZF BTB domain has been shown ␣ ͞␤ tified, ligands. The residues of the 3 4 loop have temper- to interact directly with each of mSin3A, histone deacetylase ature factors in the range of 40–60 Å2, which are among the 1, SMRT, and N-CoR (12–14). Clearly, these cannot bind highest in the entire structure. Thus, the walls of the groove simultaneously to the same site on the domain, but they may may be able to adjust their conformation to accommodate a bind to nonoverlapping regions of the dimer. We expect that putative ligand. The overall shape and symmetry of the BTB additional ligands for this and other BTB domains remain to dimer is similar to that of the HIV protease dimer (31), and be discovered, and the structure presented here provides a both share a groove that crosses the narrow dimension of the framework with which to study these interactions. molecule with a pair of diad-related aspartate residues exposed at the floor. In the protease, these are the active site residues. We thank J.-L. Couderc, F. Laski, J. Licht, C. Richardson, D. Rose, There are, however, significant differences, in that the BTB and S. Zollman for helpful discussions; I. Klitsie, D. Kuntz, and V. Ahn domain lacks the conserved Asp-Thr-Gly signature sequence for help with cloning and protein expression; and D. Thiel and of aspartate proteases, and the details of the orientation of the members of the MacCHESS staff for assistance in data collection. aspartates in the PLZF structure are not the same as in the CHESS is supported by the National Science Foundation under award protease. In addition, the PLZF BTB domain is mostly ␣-he- DMR-9311772, and the Macromolecular Diffraction at CHESS is lical, whereas aspartate proteases are built from a ␤-sheet core. supported by award RR-01646 from the National Institutes of Health. It remains to be seen whether the BTB groove is a simple C.K.E. is a postdoctoral fellow of the Deutsche Forschungsgemein- schaft. This work has been supported by grants from the Medical binding site or whether it has enzymatic activity. Research Foundation of Canada (MRCC) and the National Cancer Institute of Canada (NCIC). DISCUSSION 1. Godt, D., Couderc, J. L., Cramton, SE & Laski, F. A (1993) The hydrophobic nature of the dimer interface, the large Development (Cambridge, U.K.) 119, 799–812. interface accessible surface area, and the extensive shape 2. Zollman, S., Godt, D., Prive´,G. G., Couderc, J. L. & Laski, F. A. complimentarity (as measured by the low gap volume index of (1994) Proc. Natl. Acad. Sci. USA 91, 10717–10721. 1.44 Å) (32) are all typical of an obligate homodimer. This is 3. Bardwell, V. J. & Treisman, R. (1994) Genes Dev. 8, 1664–1677. consistent with the coupled two-state unfolding transition 4. Albagli, O., Dhordain, P., Deweindt, C., Lecocq, G. & Leprince, from a folded dimer to two unfolded monomers observed by D. (1995) Cell Growth Differ. 6, 1193–1198. differential scanning calorimetry and equilibrium denatur- 5. Koonin, E. V., Senkevich, T. G. & Cherenos, V. I (1992) Trends Biochem. Sci. 17, 213–214. ation (22). Protein domains known to form combinatorial 6. Bork, P. & Doolittle, R. (1994) J. Mol. Biol. 236, 1277–1282. hetero- generally associate either through smaller 7. Klug, A. & Schwabe, J. (1995) FASEB J. 9, 597–604. hydrophobic surfaces, as in the leucine zipper family (33), or 8. Chen, Z., Brand, N. J., Chen, A., Chen, S. J., Tong, J. H., Wang, through polar interfaces as in the SMAD protein family (34). Z. Y., Waxman, S. & Zelent, A. (1993) EMBO J. 12, 1161–1167. BTB domains have been shown by in vitro coexpression to form 9. Cook, M., Gould, A., Brand, N., Davies, J., Strutt, P., Shaknovich, specific homo- and hetero-oligomers through self-association R., Licht, J., Waxman, S., Chen, Z., Gluecksohn-Waelsch, S. et al. (3), and further work will be necessary to establish the (1995) Proc. Natl. Acad. Sci. USA 92, 2249–2253. importance of associations between different BTB domains in 10. Dong, S., Zhu, J., Reid, A., Strutt, P., Guidez, F., Zhong, H. J., vivo. Wang, Z. Y., Licht, J., Waxman, S., Chomienne, C., et al. (1996) The dimerization of transcription factors has implications Proc. Natl. Acad. Sci. USA 93, 3624–3629. 11. He, L. Z., Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., for the binding to the target DNA site, and to date, no Zelent, A. & Pandolfi, P. P. (1998) Nat. Genet. 18, 126–135. physiologically relevant DNA-binding site(s) has been re- 12. Guidez, F., Ivins, S., Zhu, J., Soderstrom, M., Waxman, S. & ported for PLZF (35). However, GAGA factor binds to high Zelent, A. (1998) Blood 91, 2634–2642. density GA repeats with a large number of potential binding 13. Hong, S. H., David, G., Wong, C. W., Dejean, A. & Privalsky, sites in close proximity, Tramtrack binds two monomer sites M. L. (1997) Proc. Natl. Acad. Sci. USA 94, 9028–9033. separated by 24 base pairs in the fushi tarazu promoter (36), 14. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H., Jr., & and ZF5 binds two sites separated by 16 base pairs in the c-myc Evans, R. M. 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