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Molecular Biology and Genetics |
The Wistar Institute [X. L., H. P., D. C. S., J. M. L-G., F. J. R. III, R. M.], Department of Chemistry [R. M.], and the Department of Biochemistry and Biophysics [X. L., R. M.], University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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homodimeric fold in which dimer interactions occur along two surfaces of the protein subunits. The conservation of BTB/POZ domain residues at the core of the protomers and at the dimer interface implies an analogous fold and dimerization mode for BTB/POZ domains from otherwise functionally unrelated proteins. Unexpectedly, the BTB/POZ domain forms dimer-dimer interactions in the crystals, suggesting a mode for higher-order protein oligomerization for BTB/POZ-mediated transcriptional repression. Biochemical characterization of PLZF-BTB/POZ harboring mutations in conserved residues involved in protein dimerization reveals that the integrity of the dimer interface is exquisitely sensitive to mutation and that dimer formation is required for wild-type levels of transcriptional repression. Interestingly, similar mutational analysis of residues within a pronounced protein cleft along the dimer interface, which had been implicated previously for interaction with corepressors, has negligible effects on dimerization or transcriptional repression. Together, these studies form a structure-function framework for understanding BTB/POZ-mediated oligomerization and transcriptional repression properties. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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gene forming the PLZF-RAR fusion protein in APL (1)
. Unlike the more common APL t(15;17) translocation forming the PML-RAR fusion protein (2
, 3)
, patients harboring the t(11;17) chromosomal translocation are resistant to treatment with pharmacological doses of RAs (4)
.
The PLZF moiety of the PLZF-RAR fusion protein contains a 120 residue BTB/POZ domain, named for its presence in the Drosophila proteins BTB (5)
, and its homology with several POZ (6)
. This domain has been found in an increasing number of proteins in poxvirus, Caenorhabditis elegans, Drosophila, and humans and is generally found at the NH2 terminus of either actin-binding or, more commonly, nuclear transcriptional regulatory proteins (7)
. Functional studies have shown that the BTB/POZ domain mediates homodimerization (6
, 8
, 9)
, heteromultimerization between different BTB/POZ-harboring proteins (6
, 10)
, and transcriptional repression in the case of several DNA regulators harboring BTB/POZ domains (10, 11, 12, 13, 14, 15, 16)
.
Much of the mechanistic detail for the function of BTB/POZ domains has come from the study of the PLZF and LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphoma 6) oncoproteins (10
, 11)
. In these cases, the BTB/POZ domain has been shown to promote transcriptional repression through the recruitment of corepressor proteins such as N-CoR and SMRT (17
, 18)
. More recently, the BTB/POZ domain of PLZF has been shown to interact with a protein complex containing N-CoR/SMRT, mSin3A, and the histone deacetylase, HDAC1, to mediate transcriptional repression (19, 20, 21)
. Moreover, this recruitment has been found to play a major role in the pathogenic effect of the PLZF-RAR fusion protein and for its resistance to treatment with RA. Specifically, a model has been proposed whereby the PLZF-RAR fusion protein acts as a potent transcriptional repressor through the ability of both the RAR (22)
and BTB/POZ moieties to recruit the SMRT/N-CoR deacetylase transcriptional repression complex (23
, 24)
. Because RA induces the release of this corepressor complex from RAR (22)
but not from PLZF, this model is consistent with the RA resistance of APL patients harboring the PLZF-RAR translocation (25
, 26)
.
Here we present the high resolution crystal structure of the BTB/POZ domain from PLZF and characterize the biochemical properties of PLZF proteins harboring site-directed mutations. The structure provides general insights into the architecture and mode of multimerization for the evolutionarily conserved BTB/POZ domain. Moreover, a correlation of the BTB/POZ domain structure with the dimerization and transcriptional repression properties of PLZF proteins harboring site-directed mutations establishes a structure-function paradigm for understanding the dimerization and transcriptional repression properties of proteins harboring BTB/POZ domains. Finally, the insights provided here provide a framework from which to design PLZF-specific inhibitory molecules that may be used to treat APL patients harboring the PLZF-RAR translocation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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SeMet-derivatized PLZF-BTB/POZ protein was prepared by growing pQE30/PLZF-BTB/POZ-transformed E. coli strain B834 (DE3; Novagen) in 4-morpholinepropanesulfonic acid-based minimal media (27)
supplemented with 50 mg/l L-SeMet and other amino acids at the suggested concentrations. Cells were grown at 28°C to an A595 of 0.4 and induced with 1 mM isoprpyl-1-thio--D-galactopyranoside to an A595 of 
1.0. The PLZF-BTB/POZ protein was isolated essentially as described for the underivatized protein. Quantitative amino acid analysis of SeMet-derivatized PLZF-BTB/POZ protein confirmed that >90% of the methionine residues had been replaced. After purification, SeMet-derivatized PLZF-BTB/POZ was concentrated to 50 mg/ml by centrifugation using a Centricon-10 microconcentrator (Amicon) in a buffer containing 40 mM Tris (pH 8.5), 100 mM NaCl, and 1 mM -mercaptoethanol and frozen as 50-µl aliquots at -70°C before crystallization. Frozen protein aliquots were thawed for use in crystallization as needed.
Crystallization and Data Collection.
Crystals of underivatized and SeMet-derivatized PLZF-BTB/POZ were prepared using 2-µl hanging drops containing 10 mg/ml PLZF-BTB/POZ, 8% isopropanol, 600 mM MgCl2, 50 mM Tris (pH 8.5), and 50 mM HEPES (pH 6.5) equilibrated over a reservoir containing two times the concentration of salts, buffer, and precipitating agent. Crystals were transiently transferred (for 5 min) to a harvest solution composed of salts, buffer, and precipitating agent at the same concentrations as the reservoir solution with the addition of 25% glycerol to facilitate X-ray data collection at cryogenic temperature (-170°C).
MAD data were collected from cryoprotected SeMet-derivatized PLZF-BTB/POZ crystals that were flash frozen in liquid propane and stored in a Dewar-containing liquid nitrogen prior to data collection at 110 K. MAD data were collected at NSLS using beamline X4A equipped with an R-AXIS IV image plate detector. The inverse-beam method was used to record Bijvoet differences from each of four different wavelengths to optimize dispersive differences: upstream remote (
1 = 0.9878 Å), the downstream remote (4 = 0.9667 Å), the inflection (2 = 0.9796 Å), and the maximum of X-ray absorption (3 = 0.9795 Å). The MAD data were processed with DENZO and SCALEPACK (Ref. 28
; Table 1
).
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). Two SeMet sites were unambiguously identified using difference Patterson maps produced with two sets of dispersive signals and three sets of Bijvoet signals, and two additional SeMet sites were confirmed using cross-difference Fourier maps using the first two sites. Phase refinement, using four SeMet sites using all Bijvoet and dispersive differences, and solvent flattening procedures were carried out with the program PHASES (29)
. The resulting electron density map was of very high quality (Fig. 1C)
and allowed for straightforward tracing of the polypeptide chain with the program O (30)
using the SeMet positions as landmarks.
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. The final model includes residues 7–122 of PLZF-BTB/POZ and 61 water molecules. Residue 6, the NH2-terminal 6xHis-tag and the COOH-terminal residue are not visible in the electron density map. A Ramachandran plot showed no residues of the protein in disallowed regions (38)
.
Site-directed Mutagenesis.
The plasmids containing the BTB/POZ domain of PLZF (residues 6–123) were constructed by PCR using the plasmid pQE30-PLZF as a template (8)
. A 5' oligonucleotide (5'-GGA TCC ACC ATG GGC ATG ATC CAG CTG CAG-3') with a BamHI site immediately 5' to a consensus Kozak sequence (ACC) at methionine 6 and a 3' oligonucleotide (5'-GAT GGA TCC CTA CTC CAG CAT CTT CAG GCA CTG-3') with a stop codon (TAG) and BamHI site after amino acid 123 were used to amplify the desired sequence. Single amino acid point mutations within the BTB/POZ domain of PLZF6–123 were created using standard PCR-mediated mutagenesis. The mutagenic primers contained the following codons: L21A, CTG to GCG; D35N, GAT to AAT; H64A, CAC to GCC; N66A, AAT to GCT; and Q68A, CAA to GCA. BamHI-digested PCR products were ligated into BamHI-digested pSP73 for in vitro translation and pM2 for in vivo expression. All PCR-derived plasmids were subjected to automated DNA sequencing of both strands to confirm the incorporation of appropriate mutations and integrity of surrounding sequences.
Gel Filtration Analysis of Wild-Type and Mutant PLZF Proteins.
Fifty µl of [35S]methionine-labeled, in vitro-translated PLZF6–123 proteins (SP6 TnT; Promega) were analyzed by gel filtration with a Superdex 200 HR 10/30 column (Pharmacia Biotech, Inc.) equilibrated in PBS (10 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.0). The column was run at 4°C at a flow rate of 0.3 ml/min, and 1-ml fractions were collected. The protein from each fraction was concentrated by deoxycholate-trichloroacetic acid precipitation (39)
. The precipitated protein was resuspended in 100 µl of 0.1 M NaOH. Thirty µl of the resuspended protein sample were resolved on a 12% Laemmli SDS-PAGE gel, and PLZF6–123 proteins were visualized by fluorography.
Transient Transfection/Luciferase Assays.
Stable expression of heterologous GAL4-PLZF6–123 fusion proteins was confirmed by transfection in COS-1 cells. One µg of a rabbit anti-GAL4 DBD polyclonal IgG (Santa Cruz Biotech) was used to detect expression of GAL4 fusion proteins by immunoprecipitation of [35S]methionine-labeled cell extracts (40)
. All transcription/luciferase assays were done in NIH/3T3 cells as described elsewhere (40)
.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Each subunit is comprised of 5 -strands and 6 -helices that associate to form a globular dimer (Figs. 1D
and 2
). The dimer has approximate dimensions of 20 Å x 30 Å x 60Å and has the appearance of a "butterfly," with each subunit forming most of each of the wings (Fig. 2, A and B)
. The strands lie on the top and bottom of each of the subunits, and the helices fill the interior and flank the sides of the dimer. There are two pronounced clefts at the top and bottom of the dimer, where the two subunits intersect (Fig. 2C)
. The smaller upper cleft has a relatively shallow groove, whereas the larger lower cleft is 20 Å long with a width and depth of 5 and 6 Å, respectively. A search through the structural database with either the monomer or dimer shows no significant homology with other protein structures, suggesting that the PLZF-BTB/POZ domain contains a novel protein fold.
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. The only exceptions are strands 1 and 5 and the NH2 terminus of helix 1, which contribute largely to dimerization (Fig. 3, B and C)
. Val36, Val37, Ile38, Met39, and Val40 from 2; Phe45 and Ala47 from 3; and Tyr70 and Leu72 from 4 form the floor of the hydrophobic core (Fig. 1C)
. The sides of the hydrophobic core are stabilized by a large number of residues in the helical segments. In particular, residues from helix 2 (Val52, Leu53, and Ala54), from helix 3 (Phe59, Leu62, and Phe63), from helix 4 (Pro77, Thr79, Phe80, Gln82, Ile83, Leu84, Tyr86, Ala87, and Tyr88), and from helix 5 (Leu99, Leu102, Leu103, Ala105, Ala106, and Leu109) make substantial contributions to the core (Figs. 1A
and 3
).
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and 3
). This conservation appears to extend beyond the subclass of BTB/POZ domain proteins that function as transcriptional regulators (7)
. In particular, His48 in the loop between 3 and 2, Leu52 from 2, Phe59 from 3, Tyr88 from 4, and Leu109 from 5 are conserved in virtually all BTB/POZ domains. Other residues that stabilize the core are also highly conserved among BTB/POZ proteins (Fig. 1B)
. Taken together, the conservation of residues that stabilize the core of the PLZF-BTB/POZ subunit strongly suggests that other BTB/POZ domains have a homologous subunit tertiary fold.
Dimerization by PLZF-BTB/POZ Implicates a Conserved Mode of Dimerization by Other BTB/POZ Domain Proteins.
Previous studies in our laboratory have used a variety of biophysical techniques to show that the BTB/POZ domain of PLZF forms a dimer with an apparent Kd < 200 nM (8)
. The structure of the PLZF-BTB/POZ dimer is consistent with the high degree of dimer stability. Overall, there are 23 residues from each subunit that contribute to dimer formation, forming a solvent excluded surface of 2400 Å2 for the dimer (Figs. 1B
and 2C
).
The principle dimer contacts between the PLZF-BTB/POZ subunits are mediated by the 1 and 5 strands and the 1 helix, which are the only secondary structural elements that do not contribute significantly to the subunit core (Fig. 3, B and C)
. The 1 strand and the NH2 terminus of the 1 helix intrude into the opposing subunit by flanking the core above (1 and 5 strands) and in the central portion of the butterfly (1 helix). For simplicity of discussion, we will refer to the symmetry-related subunit of the dimer with a primed (') designation. The 1 strand is wedged between the 5' strand and the 6' helix of the opposing subunit, making sheet interactions with the 5' strand and van der Waals and hydrogen-bond interactions with other regions of the primed subunit (Fig. 4B)
. Specifically, Ile9 makes van der Waals interactions with Leu99 of 5', Ala96' in the loop between 5' and 6' and Met121 of 6'; Ile11 makes van der Waals interactions with Tyr113, Leu114, Glu117, and Cys118 of helix 6'; and the N
2 of Asn13 hydrogen bonds to the side-chain hydroxyl of Tyr86 in helix 4'.
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1 helix and the proceeding loop is situated between the 1' and 2' helices and makes mostly van der Waals interactions with residues within these helices as well as with the 3' and 4' helices (Fig. 3C)
. Specifically, 1-helix residues His16, Pro17, Leu20, Leu21, and Met27 make a series of van der Waals interactions with residues from helices 1', 2', 3', and 4'. In particular, Leu21 from 1', Cys54 from 2', and Ala90 from the loop between 4' and 5' play important roles in stabilizing the dimer. Thr32, Leu33, and Asp35 in the loop proceeding helix 1 also contribute to dimer stability. His64 from 3' plays a particularly important role in this regard.
Nearly 80% of the residues that stabilize the PLZF-BTB/POZ dimer show conservation within the BTB/POZ domain family (Fig. 1A)
. In particular, His16 and Leu21 in helix 1 and Asp35 in the loop proceeding the 1 helix are highly conserved and play important roles in dimer stability. All but the aspartic acid play a hydrophobic role in dimer stability. Asp35, in contrast, makes a direct hydrogen bond to Arg49 (moderately conserved within the BTB/POZ family) and a water-mediated hydrogen bond to Asp35'. Taken together, the degree of conservation within residues that stabilize dimer formation suggests that BTB/POZ domains from otherwise unrelated proteins will form dimers with similar quaternary arrangements. Correlating well with our findings, there are several BTB/POZ domains that have been shown to form homodimers. Among them are PLZF (8)
, ZID (6)
, Ttk (6)
, bab (9)
, and BAZF (11)
. Indeed, the BTB/POZ domain appears to be an ideally suited dimerization module.
Dimer-Dimer Interactions in the Crystals Implicates a Propensity for Higher Order Oligomerization by BTB/POZ Domains in Vivo.
Comparison of the PLZF-BTB/POZ structure derived here with that of the recently published PLZF-BTB/POZ structure determined by Ahmad et al. (42)
shows a high degree of structural similarity between the protein dimers with an RMS deviation between all atoms of 1.1 Å2. Strikingly, this comparison also shows that although the two structures were obtained in different crystal lattice environments, both show structurally homologous dimer-dimer interactions in the crystal lattice (Fig. 4A)
. These dimer-dimer contacts bury a total of 1200 Å2 of solvent excluded surface and is largely mediated by 2 4-stranded antiparallel -sheet involving 1 and 5' from one dimer with the corresponding segments of the symmetry related dimer. In addition, Ala96 in the loop between 5 and 5 makes a van der Waals contact with Met121 at the COOH-terminus of 6 in the symmetry related dimer. The 6-mediated interactions at the dimer-dimer interface are somewhat more extensive in the PLZF-BTB/POZ structure described by Ahmad et al. (42)
because their 6 helix contains an additional turn of secondary structure. It is interesting to note that the mode of dimer-dimer interaction observed in the crystals does not prohibit the formation of extended dimer-dimer interactions, which would result in the formation of even higher order multimers (Fig. 4, B and C)
. Taken together, these observations suggest that the BTB/POZ domain may mediate the formation of higher-order multimers in vivo.
Correlating well with our findings, two recent studies have shown that the BTB/POZ domain of the GAGA transcription factor directly mediates the formation of higher-order oligomers to bind multiple GAGA sites that are found in natural target promoters in vivo (41 , 43) . Moreover, the formation of these higher-order oligomers have been shown to be correlated with the cooperative nature of GAGA transcription factor binding to multiple DNA-binding sites and correlated with the finding that this cooperativity is strictly dependent on the presence of the GAGA BTB/POZ domain. Interestingly, natural GAGA promoters display a large degree of variability between GAGA sites, also correlating well with the relatively flexible dimer-dimer interactions seen in the crystals.
Our findings of higher-order BTB/POZ oligomers may also explain other studies that find that the BTB/POZ domains from some proteins mediate specific hetero-oligomers. For example, Ttk can form oligomers with itself and with the GAGA protein but not with the BTB/POZ region of ZID (6) . In addition, BAZF has been shown to form oligomers with itself and with BCL6 (11) . Taken together, the BTB/POZ domain appears to be an ideally suited module for both homo and hetero protein multerimerization.
Biochemical Analysis of PLZF Proteins Harboring Site-directed Mutations Shows That Dimerization Is Required for Transcriptional Repression.
The recent study by Ahmad et al. (42)
has suggested that a pronounced cleft along the bottom of the PLZF dimer (Fig. 2C)
may be a site of corepressor interaction. To directly test this hypothesis and to directly probe the functional significance of dimer formation by PLZF, we carried out site-directed mutagenesis coupled with biochemical analysis of these mutant proteins. We prepared five different site-directed mutations that fell into two subgroups (Fig. 2C)
. The first two (L21A and D35N) involved residues that play critical roles in dimerization; and the second group (H64A, N66A, and Q68A) involved residues in the pronounced cleft at the base of the PLZF-BTB/POZ domain dimer. Each of the mutants were tested for both dimerization and transcriptional repression properties. To evaluate the effects of these mutations on the dimerization of the BTB/POZ domain, the wild-type PLZF6–123 and each mutant described herein were in vitro translated and then subjected to gel filtration (Fig. 5)
. Consistent with expectations, the wild-type PLZF6–123 protein, as well as each of the proteins harboring mutations in the cleft region, eluted from the sizing column at a molecular weight consistent with a dimeric PLZF-BTB/POZ species. In addition, proteins harboring mutations in the dimerization interface of PLZF-BTB/POZ eluted in two peaks, one near the void volume (Mr 
670,000), indicative of protein aggregates and another at a position consistent with a monomeric PLZF-BTB/POZ domain (Mr 17,000). These results indicated that the L21A and D35N mutant proteins were defective in dimer formation.
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. Each fusion protein demonstrated stable and comparable nuclear-localized expression in COS-1 cells (Fig. 6B)
, suggesting comparable DNA-binding properties for each of our protein fusions. The wild-type GAL4-PLZF6–123 BTB/POZ domain and each mutant version thereof were cotransfected with 2 TK-luciferase reporter downstream of four copies of the GAL4 consensus UAS in NIH/3T3 cells (Fig. 6A)
. The wild-type GAL4-PLZF6–123 protein demonstrated significant repression of the luciferase reporter gene activity (Fig. 6C)
. Expression of the of the D35N mutant protein, which fails to dimerize properly (Fig. 5B)
, demonstrated a significantly impaired repression function (Fig. 6C)
. To a lesser extent, the repression function of the L21A mutant protein was also impaired (Fig. 6C)
. All other mutants (H64A, N66A, and Q68A) demonstrated negligible effects on the repression function of the BTB/POZ domain of PLZF (Fig. 6C)
. Significantly, these data suggest that the repression function of the PLZF-BTB/POZ domain is strictly dependent upon homodimerization. Moreover, these data suggest that the cleft region of the PLZF-BTB/POZ domain may not play a significant role in the transcriptonal repression property of PLZF.
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and 3C
). An asparagine to histidine change at position 25 (using PLZF numbering) did not have a detectable effect on N-CoR and SMRT interaction or transcriptional repression. The structure of the PLZF-BTB/POZ domain shows that Asp25 is packed on the interior of the protein, thus playing an important role in protein folding (Fig. 3A)
. Modeling of a histidine in position 25 shows that it could carry out a comparable role in BTB/POZ domain stability. Taken together, our results and the results of Huynh and Bardwell (10)
underscore the importance of BTB/POZ domain dimerization for N-CoR and SMRT-mediated transcriptional repression.
The precise mode of corepressor recognition by PLZF must await the structure of an appropriate PLZF-BTB/POZ/corepressor complex. Nonetheless, the structural and functional information provided here provides a framework from which to use structure/function analysis to better understand the N-CoR and SMRT binding and repression properties of the BTB/POZ domain of PLZF. Moreover, the information provided here provides a conceptual and structural scaffold from which to design PLZF-specific inhibitory molecules that may target the dimerization and/or interaction of PLZF with corepressor proteins to be used to treat APL patients harboring the PLZF-RAR translocation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Leukemia Research Foundation (to R. M.) and NIH Basic Cancer Research Training Grant CA09171 (to D. C. S). F. J. R. was supported in part by NIH Grant CA52009, Core Grant CA10815, Core Grant DK50306, and Grants DK49210, GM54220, DAMD17-96-1-6141, and ACS NP-954; the Irving A. Hansen Memorial Foundation; the Mary A. Rumsey Memorial Foundation; and the Pew Scholars Program in the Biomedical Sciences. 
2 Present address: Childrens Hospital and Medical Center, Seattle, WA 98105.
3 To whom requests for reprints should be addressed, at The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-5006; Fax: (215) 898-0381; E-mail: marmor{at}wistar.upenn.edu
4 The abbreviations used are: PLZF, promyelocytic leukemia zinc finger; RAR, retinoic acid receptor; APL, acute promyelocytic leukemia; RA, retinoic acid; BTB, Broad complex, Trametrack, and Bric a brac; POZ, poxvirus and zinc finger proteins; SMRT, silencing mediator of retinoid and thyroid receptor; SeMet, selenomethionine; MAD, multiwavelength anomalous dispersion.
Received 6/14/99. Accepted 8/19/99.
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REFERENCES |
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locus due to a variant t(11;17) translocation associated with acute promyelocytic leukemia. EMBO J., 12: 1161-1167, 1993.[Medline]
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A. Melnick, K. F. Ahmad, S. Arai, A. Polinger, H. Ball, K. L. Borden, G. W. Carlile, G. G. Prive, and J. D. Licht In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions Mol. Cell. Biol., September 1, 2000; 20(17): 6550 - 6567. [Abstract] [Full Text]
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 S. A. Mackler, L. Korutla, X.-Y. Cha, M. J. Koebbe, K. M. Fournier, M. S. Bowers, and P. W. Kalivas NAC-1 Is a Brain POZ/BTB Protein That Can Prevent Cocaine-Induced Sensitization in the Rat J. Neurosci., August 15, 2000; 20(16): 6210 - 6217. [Abstract] [Full Text] [PDF]
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K. D. Huynh, W. Fischle, E. Verdin, and V. J. Bardwell BCoR, a novel corepressor involved in BCL-6 repression Genes & Dev., July 15, 2000; 14(14): 1810 - 1823. [Abstract] [Full Text]
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C. Schumacher, H. Wang, C. Honer, W. Ding, J. Koehn, Q. Lawrence, C. M. Coulis, L. L. Wang, D. Ballinger, B. R. Bowen, et al. The SCAN Domain Mediates Selective Oligomerization J. Biol. Chem., May 26, 2000; 275(22): 17173 - 17179. [Abstract] [Full Text] [PDF]
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, residues that are buried upon dimer formation. C, experimental MAD electron density map after solvent flattening at a resolution of 2.0 Å showing 
