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Originally published In Press as doi:10.1074/jbc.M601980200 on April 28, 2006

J. Biol. Chem., Vol. 281, Issue 26, 18208-18215, June 30, 2006
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The Carboxyl-terminal Nucleoplasmic Region of MAN1 Exhibits a DNA Binding Winged Helix Domain*

Sandrine Caputo{ddagger}1, Joël Couprie{ddagger}1, Isabelle Duband-Goulet§1, Emilie Kondé{ddagger}, Feng Lin2, Sandrine Braud{ddagger}, Muriel Gondry{ddagger}, Bernard Gilquin{ddagger}1, Howard J. Worman3, and Sophie Zinn-Justin{ddagger}14

From the {ddagger}Département d'Ingénierie et d'Etudes des Protéines/Direction des Sciences du Vivant, Bâtiment 152, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette Cedex, France, §Institut Jacques Monod-CNRS Unité Mixte de Recherche 7592, Universités Paris 6/7, 2 Place Jussieu, 75251 Paris Cedex 05, France, and Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received for publication, March 1, 2006 , and in revised form, April 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The nuclear envelope separates the nucleus from the cytoplasm in eukaryotic cells. It consists of inner and outer nuclear membranes and nuclear pore complexes. The inner nuclear membrane is closely associated with the underlying chromatin and nuclear lamina. For many years, the nuclear envelope was thought to function mainly as an architectural stabilizer of the nucleus, participating in assembly and disassembly processes during mitosis. However, recent findings demonstrate that nuclear envelope proteins are involved in fundamental nuclear functions, such as chromatin organization and gene expression (1). Inherited or de novo mutations in genes encoding nuclear envelope proteins cause a wide range of human diseases (2). These findings emphasize the importance of understanding the functions of the nuclear envelope in both physiologic and pathologic states.

MAN1 (also known as LEMD3) is a transmembrane protein of the inner nuclear membrane. It was originally identified as an antigen recognized by self-antibodies from the serum of a patient with a collagen vascular disease (3, 4). MAN1 is part of a protein complex essential for chromatin organization and cell division. It is analogous to the yeast protein SRC1, which may play a role in sister chromatid separation (5). In Xenopus embryos, overexpressed MAN1 induces the formation of a secondary neural axis by binding directly to the MH2 domain of Smad1, Smad5, or Smad8, thus antagonizing bone morphogenetic protein signaling (6, 7). Similarly, in humans MAN1 binds to the MH2 domain of the R-Smads Smad1, Smad2, and Smad3, which mediate signaling by activin, bone morphogenic protein, and transforming growth factor beta (8, 9). Heterozygous loss-of-function mutations in the human gene encoding MAN1 that disrupt this critical interaction cause sclerosing bone dysplasias characterized by increased bone density and sometimes skin abnormalities (10). Thus, mutations in a ubiquitous nuclear envelope protein give rise to relatively tissue-specific disease phenotypes, suggesting a role for MAN1 in the regulation of tissue-specific gene transcription, as has similarly been proposed for nuclear lamins (11).

Sequence analysis indicates that MAN1 spans the inner nuclear membrane twice, resulting in a protein with amino-terminal and carboxyl-terminal nucleoplasmic domains (4). At its amino terminus, MAN1 contains a LEM domain that is present in several proteins, including the inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin (4, 12-14). This domain interacts with the DNA and chromatin-binding protein Barrier-to-Autointegration Factor (15, 16). The entire amino-terminal nucleoplasmic region of MAN1 also binds to the nuclear intermediate filaments lamin A and lamin B1 and to emerin (17). Thus, it mediates protein-protein interactions through contacts with the chromatin and the nuclear lamina. This domain is also necessary for efficient localization of MAN1 in the inner nuclear membrane (18).

Here we have examined the three-dimensional structure of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for the inhibition of physiologically important signaling pathways through an interaction with several R-Smads (8, 9). This region of MAN1 has also been shown to bind to Barrier-to-Autointegration Factor and to the transcriptional regulators GCL and Btf (17). It comprises a first fragment (amino acids 655-758) showing sequence characteristics of a globular domain and a second fragment (amino acids 782-911) predicted to be an RRM-like protein interaction domain named U2AF homology motif (UHM)5 (19). We report that the first globular domain adopts a three-dimensional structure generally found in DNA binding regions of transcription factors. We show that indeed the entire carboxyl-terminal region of MAN1 is involved in DNA binding and propose that this interaction is synergetic to the binding of MAN1 to different transcriptional regulators, particularly R-Smads.


Figure 1
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  		FIGURE 1.
Superimposition of 1H-15N HSQC spectra of MAN1CA and MAN1CB. Overlay of the 1H-15N HSQC spectra obtained at 300 K for MAN1CA (blue) and for MAN1CB (red). Both protein fragments were prepared in 50 mM phosphate/Tris buffer and 150 mM NaCl at pH 6.0.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sample Preparation—All cloning procedures were performed according to standard methods (20). The region from amino acid 655 to amino acid 775 of MAN1 (MAN1CA) was overexpressed in Escherichia coli strain BL21 DE3 pLys S transformed with a construct generated in pGEX-4T-1 (Amersham Biosciences, Inc.) that encodes glutathione S-transferase and a thrombin cleavage site fused to MAN1CA. The fusion protein was purified using glutathione-Sepharose 4B (Amersham Biosciences) and cleaved using thrombin protease. Because of the cloning strategy, the peptide resulting from the cleavage comprises additional residues from 1 to 5, MAN1 residues from 6 to 126, and again additional residues from 127 to 133. Uniformly labeled 15N protein was produced in minimum medium M9 containing 1 g·liter-1 of (15NH4)2SO4 (Boehringer) as the nitrogen source. Uniformly labeled 13C/15N protein was produced in a rich medium prepared from uniformly labeled 13C/15N Spirulina maxima cyanobacteria. The resulting protein was characterized by electrospray ionization mass spectroscopy and amino terminus sequencing. NMR samples (~0.8 mM) were prepared in 50 mm phosphate buffer (pH 6.0) containing 150 mM NaCl in either 90% H2O/10% D2O or in 100% D2O, 1 mM EDTA, a protease inhibitor mixture (Sigma-Aldrich), 1 mM Tris(2-chloroethyl) phosphate, and 1 mM NaN3. 3-(Trimethylsilyl)[2,2,3,3-2H4] propionate (TSP) was added as a chemical shift reference. 13C and 15N chemical shifts were referenced indirectly to TSP, using the absolute frequency ratios.

The region of MAN1 from amino acid 658 to amino acid 910 (MAN1CB) was overexpressed in E. coli strain BL21 DE3 Star transformed with a plasmid that encodes ZZ fusion, a cleavage site for tobacco etch virus, protease and MAN1CB (21). It was purified using immunoglobulin IgG-SepharoseTM 6 Fast Flow (Amersham Biosciences) and cleaved using the catalytic domain of tobacco etch virus protease. Uniformly labeled 15N protein was produced in minimum medium M9 containing 1 g·liter-1 of (15NH4)2SO4 (Boehringer) as the nitrogen source. The resulting protein was characterized by electrospray ionization mass spectroscopy and amino terminus sequencing. NMR samples of the protein at ~0.5 mM were prepared in 50 mM Tris buffer (pH 6.0) containing 150 mM NaCl in either 90% H2O/10% D2O or in 100% D2O, 1 mM EDTA, a protease inhibitor mixture (Sigma-Aldrich), 1 mM Tris(2-chloroethyl) phosphate, and 1 mM NaN3. TSP was added as a chemical shift reference. 15N chemical shifts were referenced indirectly to TSP, using the absolute frequency ratios. MAN1CA and MAN1CB mutants were generated with QuikChange multisite-directed mutagenesis kit (Stratagene) following the manufacturer's instructions.

NMR Spectroscopy—All assignment experiments of MAN1CA were performed at 30 °C on Bruker DRX-600 or DRX-900 spectrometers equipped with a triple resonance probe according to the previously reported procedure (22). The nuclear Overhauser effect cross-peak volumes used for structure calculation were measured on five NOESY experiments (a 15N-HSQC-NOESY in H2O and a 13C-HSQC-NOESY in D2O recorded at 900 MHz with a 100-ms mixing time at the European Large Scale Facilities in Utrecht, Netherlands, a 15N-HSQC-NOESY in H2O with a 150-ms mixing time and a 13C-HSQC-NOESY in D2O with a 200-ms mixing time and a 13C-HSQC-NOESY in the 13C aromatic region with a 200-ms mixing time, all three recorded on a local 600-MHz spectrometer equipped with a triple resonance TXI cryoprobe). {phi} torsion angle values were deduced from the analysis of the Hn-Ha and HMQC-J experiments (23, 24). Hydrogen-bound restraints were derived from slowly exchanging amide protons, identified by measuring the amide proton exchange rates from 1H-15N HSQC spectra recorded at different times on a protein sample dissolved in D2O. All spectra were processed with the programs Xwinnmr (Bruker) or NMRPipe (25) and analyzed using Felix (Accelrys).

Solution Structure Determination—We solved the three-dimensional solution structure of the carboxyl-terminal domain of MAN1CA using heteronuclear double and triple resonance NMR spectroscopy and molecular modeling. Coordinates and NMR restraints were deposited at the Protein Data Bank. The solution structure was calculated on the basis of the analysis of 3581 nuclear Overhauser effect cross-peaks (765 were picked on the 15N-HSQC-NOESY 600 MHz, 756 on the 15N-HSQC-NOESY 900 MHz, 1241 on the 13C-HSQC-NOESY 900 MHz, 763 on the 13C-HSQC-NOESY 600 MHz, and 56 on the 13C-HSQC-NOESY aromatic 600 MHz, respectively). A semiautomated iterative assignment procedure was applied for the assignment and the construction of the three-dimensional structures (26). A force field adapted to NMR structure calculation (file parallhdg.pro in CNS 1.0) (27) was used. On this basis, 1850 restraints were generated. Thus, the mean number of distance restraints/residue yields 19.2 for the segment Arg-6 to Ile-111. Furthermore, 169 couples of ({phi}, {Psi}) torsion angles were derived either from the backbone 1H, 15N, and 13C chemical shifts using the program TALOS (28) or from the Hn-Ha and HMQC-J data. Finally, 8 hydrogen bonds were imposed during the structure calculation. At the last step, 1000 structures were calculated and the 20 best structures were selected and refined with a standard energy function (CHARMM22), including an electrostatic energy term. This term was calculated with no net charge on the side-chain atoms and with a distance-gated dielectric constant. Analysis of the 20 final structures showed that no distance violations larger than 0.5 Å were present and that the covalent geometry was respected. The r.m.s.d. around the average structure was 1.0 Å for the backbone and 1.6 Å for the heavy atoms.

DNA Preparation—The 211-base pair DNA fragment was generated by polymerase chain reaction with a thermostable DNA polymerase (Promega) using a PTC-100 PCR System (MJ Research, Inc.). The 211-base pair DNA fragment, obtained from the DraI and BamHI double digest of the plasmid pUC(357.4), was used as template and the 5'-AAATAGCTTAACTTTCATCAAGCAAG-3' and 5'-CCCGGGCGAGCTCGAATTCC-3' oligonucleotides as sense and antisense primers. 5'-end labeling with [32P]ATP and T4 polynucleotide kinase was performed according to standard protocols (20).

Protein-DNA Interactions—Proteins were diluted to the concentrations indicated in the Fig. 5 legend in 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 mM Tris(2-chloroethyl) phosphate, 50 mM NaCl, and 0.1% Triton X-100. They were incubated with the radioactive 211-base pair DNA fragment for 3 h at room temperature. Protein-DNA complexes were analyzed on 5% polyacrylamide gels at an acrylamide to bis-acrylamide ratio of 29/1 (w/w) in 12.5 mM Tris-HCl, pH 8.4, 95 mM glycine, and 0.5 mM EDTA. After 1 h of pre-electrophoresis, samples were loaded onto the gels and resolved by electrophoresis at 70 V for 2 h. DNA was detected by auto-radiography of dried polyacrylamide gels at -80 °C using Biomax MR films (Kodak) and an intensifying screen. For affinity measurements, dried polyacrylamide gels were exposed to a phosphor screen, and measurements of the radioactive signals were performed with a STORM 860 scanner (Amersham Biosciences) using ImageQuant software (GE Healthcare).

Molecular Modeling of the MAN1 Winged Helix-DNA Complex— The winged helix domain of MAN1 was superimposed onto the winged helix domain of MecI-DNA complex using the program Sybyl 6.9. The superimposition was done by fitting the C{alpha} atoms of the following segments 6-23, 24-25, 26-37, 38-53, 60-64, and 65-68 of MecI on 15-32, 44-45, 46-57, 60-75, 82-86, and 93-96 of MAN1CA (resulting r.m.s.d. 3.4 Å). These segments correspond to the secondary elements of the winged helix motif. Then, in the MecI-DNA complex, the winged helix domain of MecI was replaced by the corresponding domain of MAN1. To remove the small number of steric clashes, 1000 steps of steepest descents energy minimization were run using the program CHARMM (29). Side-chain conformations were allowed to vary only on the MAN1 winged helix domain, keeping the entire DNA fixed and maintaining the MAN1 winged helix domain backbone by progressively decreasing harmonics constraints. A MAN1 winged helix-DNA complex model with no bad clashes and reasonable interaction energy was thus obtained.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The MAN1 Carboxyl-terminal Nucleoplasmic Region Contains a Well Structured Domain—The carboxyl-terminal nucleoplasmic region of MAN1, comprising residues 655-910, exhibits two potential globular domains. The region from amino acid 655 to 758 is conserved in all MAN1 analogs (called MAN1 and LEM2 proteins) (30) and shows globular domain sequence characteristics (31); the region between amino acids 785 and 910 is found only in MAN1 proteins and shows an unusual RRM motif called UHM, predicted to adopt an {alpha}/beta fold and to be involved in protein recognition (19). We have recorded the 1H-15N HSQC spectra of MAN1CA (aminos acid 655-775 of MAN1) and MAN1CB (amino acids 658-910), corresponding to the first predicted globular domain and the entire carboxyl-terminal nucleoplasmic region of MAN1, respectively. The spectrum of MAN1CA is well dispersed, confirming that the region from amino acid 655 to 775 adopts a globular structure in solution. Superposition of MAN1CA and MAN1CB spectra shows that all peaks of MAN1CA are found at identical chemical shifts in MAN1CB spectrum (Fig. 1). This suggests that the three-dimensional structure of MAN1CA is not affected by the presence of the putative UHM domain. Furthermore, only ~40 additional peaks are found on the MAN1CB spectrum compared with the MAN1CA spectrum. These peaks are mostly clustered between 7.5 and 8.5 ppm in the proton dimension. This is not consistent with the presence of a well structured {alpha}/beta UHM domain. A slow proteolysis of MAN1CB is observed at 300 K, and analysis of the resulting peptides by SDS-PAGE and amino-terminal sequencing revealed that only MAN1CA is resistant to proteolysis. Finally, expression in E. coli of the second putative domain alone (amino acid 776-910) yielded a rapidly aggregating protein. All these data suggest that only the first globular domain adopts a stable three-dimensional structure on the NMR time scale (millisecond).


Figure 2
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  		FIGURE 2.
Three-dimensional structure of the MAN1 region (666-750). Schematic representation of residues 666-750. The secondary structures are colored in green for {alpha}-helix H1, magenta for {alpha}-helix H2, red for {alpha}-helix H3, and yellow for the three beta-strands.

 
The Fragment from Amino Acid 666 to Amino Acid 750 of MAN1 Adopts a Well Defined {alpha}/beta Structure—The three-dimensional structure of MAN1CA was characterized using heteronuclear NMR. In the following, residues belonging to this fragment are numbered from 6 to 126 (residues 1-5 and 127-133 are additional amino acids linked to the plasmid construction). Backbone and side-chain 1H, 13C, and 15N resonance assignments were performed from residue 6 to 112 (22). Next, molecular modeling calculations were carried out to obtain a structure consistent with the 1811 NOESY-derived proton-proton distances and the 169 dihedral angle values deduced from TALOS (Table 1). The region between residues 17 and 101 (amino acids 666-750, using the MAN1 numbering) adopts a well defined {alpha}/beta fold. The backbone r.m.s.d. calculated on this fragment with respect to the mean coordinate yields 1.0 Å. The three-dimensional structure of the region between residues 17 and 101 is constituted of three {alpha}-helices, H1 (residues 17-37), H2 (residues 47-54), and H3 (residues 58-76), and three beta-strands, S1 (residues 44-46), S2 (residues 81-88), and S3 (residues 92-99), organized into a H1-S1-H2-H3-S2-S3 topology (Fig. 2). Thus, the amino-terminal half of the domain is mainly {alpha}-helical, whereas the carboxyl-terminal half is composed of two large beta-strands arranged in a twisted anti-parallel beta-sheet.


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  		TABLE 1
Structural statistics for the human MAN1 655–775 fragment The van der Waals energy is calculated with a Lennard-Jones potential. The electrostatic energy is calculated with no net charge on side-chain atoms and a distance-gated dielectric constant. CHARMM22 parameters are used. NOE, nuclear Overhauser effect.

 
The helices form a three-helix bundle. They are amphipathic, and their hydrophobic core is constituted by Met-20, Val-21, Ile-24, Ile-25, Val-27, and Leu-28 (H1), Val-50, Leu-54 (H2), and Trp-67, Ala-70, Val-71, Leu-74 (H3). The three-stranded beta-sheet is packed onto the three-helix bundle. The {alpha}/beta interface is mainly hydrophobic. It is constituted by Leu-28 (H1), Ile-47 and Val-50 (H2), Trp-67 and Val-71 (H3), which contact the hydrophobic face of the three-stranded beta-sheet composed of Met-45 (S1), Val-81 (S2), and Trp-96, Trp-98 (S3). This interface also involves a hydrogen bond network between the backbone of Leu-28 (H1), the side chain of Asn-32 (H1), the side chain of Trp-98 (S3), and the backbone of Arg-80 (S2).

The {alpha}/beta Structure of MAN1 Corresponds to a Winged Helix Domain—The three-dimensional structure of the fragment between residues 17 and 101 of MAN1CA was submitted to the DALI server. Its structure is close to the three-dimensional structure of numerous proteins belonging to the winged helix superfamily as defined by SCOP. Winged helix domains are mainly used for DNA recognition (32). The winged helix domain of MAN1 is structurally similar to several DNA binding domains belonging to transcription factors (PDB code 1OKR [PDB] , Z-score 4.0, sequence identity 5%, Fig. 3A; 1P4X, Z-score 3.8, sequence identity 9%) and to histone H5 (1HST, Z-score 3.5, sequence identity 9%). However, recently winged helix domains were also described as protein-RNA (19, 33, 34) and protein-protein (4, 35) interaction modules. The MAN1 winged helix domain is also structurally similar to a protein-protein interaction module belonging to the ESCTR-II endosomal trafficking complex (1U5T; Z-score 4.0, sequence identity 5%, Fig. 3B). In this structure, contacts between different winged helices are mediated on one side by the concave surface formed by helices H2, H3, and beta-sheet S2-S3 and on the other side by the loop connecting H3 to S2.


Figure 3
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  		FIGURE 3.
Superimposition of the WH domain of MAN1 ({alpha}-helices in blue and beta-strands in yellow) with those of a domain of the bacterial transcriptional repressor MecI (A) (PDB code 1OKR, {alpha}-helices in cyan and beta-strands in red) and a module of the ESCTR-II endosomal trafficking complex (B) (PDB code 1U5T, {alpha}-helices in cyan and beta-strands in red).

 


Figure 4
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  		FIGURE 4.
Electrostatic properties at the surface of the MAN1 WH domain. A, surface representation of the electrostatic potential at the surface of the MAN1 WH domain (from positively charged, in blue, to negatively charged, in red). B, schematic representation of the backbone (in gray) and the positively charged side chains (in blue) of the MAN1 WH domain.

 


Figure 5
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  		FIGURE 5.
Comparative DNA binding of MAN1CA and MAN1CB to DNA. A, increasing concentrations of MAN1CA wild-type and mutant peptides were incubated with a 211-bp DNA fragment at a concentration of 18 nM in 50 mM NaCl: 10-fold (lanes 2, 6, 10, and 14), 20-fold (lanes 3, 7, 11, and 15), 40-fold (lanes 4, 8, 12, and 16), and 80-fold (lanes 5, 9, 13, and 17) molar excess of MAN1CA (655-775). Lane 1 indicates the mobility of naked DNA. B, increasing concentrations of MAN1CB wild-type and mutant peptides were incubated with a 211-bp DNA fragment at a concentration of 18 nM in 50 mM NaCl: 5-fold (lanes 2, 6, 10, and 14), 10-fold (lanes 3, 7, 11, and 15), 20-fold (lanes 4, 8, 12, and 16), and 40-fold (lanes 5, 9, 13, and 17) molar excess of MAN1CB (658-910). Lane 1 indicates the mobility of naked DNA.

 
Both the Winged Helix Domain and the Entire Carboxyl-terminal Nucleoplasmic Region of MAN1 Interact with DNA—Calculation of the electrostatic potential at the surface of the MAN1 winged helix domain shows that the amino-terminal region of H3 and the tip of the beta-sheet are mainly positively charged (Fig. 4). In particular, the sequence RKKMKKVWDR found in H3, which corresponds to the recognition helix in known complexes of winged helix domains with DNA (32), presents 6 positively charged residues (Fig. 4B). We tested the importance of these 6 residues for the binding of MAN1CA and MAN1CB to DNA by gel shift retardation assay. Therefore, we produced three types of mutants R60A/K61A/K62A, K64A/K65A, and R69A of the recognition helix H3. Interaction with a 211-base pair linear DNA fragment was tested by electrophoresis in a 5% polyacrylamide gel. Fig. 5 shows that the DNA forms complexes with wild-type proteins as demonstrated by delayed migration compared with naked DNA (Fig. 5, A and B, lanes 2-5). The apparent affinities of the two peptides for DNA are 150 ± 13 nM for MAN1CA and 50 ± 18 nM for MAN1CB. The appearance of discrete bands with MAN1CB as well as the higher affinity of MAN1CB for DNA suggests that the second domain may play a role in the stability of the complex. Consistently, for all mutants of MAN1CA a complete loss of DNA binding is observed (Fig. 5A, lanes 6-17), whereas the mutant of MAN1CB, which exhibits a single mutation, R69A, shows only a 10-fold decrease of affinity for DNA (Fig. 5B, lanes 14-17). Yet the two mutants (R60A,K61A,K62A and K64A,K65A) of MAN1CB present a complete loss of DNA binding (Fig. 5B, lanes 6-13). Thus, several positively charged residues of the recognition helix H3 are involved in the binding of the carboxyl-terminal region of MAN1 to DNA. The poorly structured UHM domain also contributes to the affinity of MAN1 for DNA.


Figure 6
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  		FIGURE 6.
Modeling the complex between the WH domain of MAN1 and DNA. A, structural alignment of {alpha}-helices H3 of winged helix domains binding to B-DNA (PDB code is indicated in parentheses). Black boxes indicate the limits of H3 sequences. Underlined residues contact DNA through hydrogen bonds; residues in bold interact with bases. The central arginine residue is boxed in a red square; on both sides of this arginine, blue and orange boxes surround hydrophobic and hydrophilic residues, respectively. B, model of the complex between the WH domain of MAN1 and DNA based on analysis of the MecI-DNA complex (1SAX).

 
Modeling of the Complex of MAN1 Winged Helix Domain with DNA— Nine crystal structures of winged helix domains in complex with B-DNA are available. We analyzed these structures to get insight into the structural determinants of the DNA recognition by the winged helix motif. Fig. 6A shows the structural alignment of the recognition helix residues in these complexes. Interestingly, the arginine residue that makes multiple hydrogen bonds with a guanine base is roughly at the center of helix H3. This residue is flanked on both sides by hydrophobic residues. Before and after these two hydrophobic regions, several hydrophilic residues of the H3 helix make contacts with DNA. The MAN1 sequence presents all these characteristics: a central Arg-69, flanked by two hydrophobic regions (Val-66-Trp-67 and Ala-70-Val-71) with hydrophilic residues on both sides. To construct a structural model of the complex between the MAN1 winged helix domain and DNA, we selected the MecI-DNA complex (36) because of the high DALI score found for MecI (PDB code 1OKR [PDB] ). Our domain was globally fitted onto the winged helix structure of MecI in complex with DNA using the DALI alignment, and Arg-69 of MAN1 was adjusted to Arg-51 of MecI. In such a model, the winged helix domain of MAN1 is in close contact with the DNA fragment (Fig. 6B). In particular, the side chain of Arg-69 interacts with the Gua0 (our DNA reference point) by two H-bonds. Moreover, at the beginning of helix H3, Lys-62 and Lys-65 form a salt bridge with the backbone phosphates at Ade+2 and Ade+3, respectively. Therefore, in this complex, 1 residue of each of 3 mutated segments of MAN1 interacts with DNA. Finally, as in other complexes of winged helix domains with DNA, the wing1 (the turn between beta2 and beta3) of the winged helix of MAN1 makes contact with the minor groove: Phe-93 at the tip of the beta-sheet is positioned in this groove. Thus, the calculated model is consistent with our experimental data as well as with the published structures of winged helix transcription factors bound to DNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The Carboxyl-terminal Nucleoplasmic Region of MAN1 Contains a Well Folded Winged Helix Domain but Does Not Show the Predicted Stable UHM Domain—NMR analysis of the solution structure of the carboxyl-terminal nucleoplasmic region of MAN1 showed that this region is composed of a DNA binding winged helix domain followed by a poorly stable/folded peptide fragment. Superimposition of the one-dimensional 1H NMR spectra of MAN1CA and MAN1CB shows that the two spectra are similar (data not shown). The low contribution of the second putative UHM domain to the one-dimensional 1H NMR spectrum of MAN1CB indicates that this domain is not completely disordered. Gel filtration experiments carried out on MAN1CB do not suggest extensive oligomerization or aggregation of this fragment, which could be responsible for the lack of NMR signal. More likely, this lack of signal could be a consequence of a partial unfolding or conformational exchange process. However, the carboxyl-terminal nucleoplasmic region of MAN1 is at least partially functional in the conditions used in this study, as it is capable of binding to the MH2 domain of Smad2 and Smad3 (data not shown). Within the three structurally characterized UHM domains, two of them (from proteins U2AF35 (37) and U2AF65 (38)) are in complex with a peptide ligand. Furthermore, the UHM domain of U2AF35 adopts its stable three-dimensional structure upon binding to its ligand (39). The third domain, belonging to the protein TgDRE (40), was characterized alone and was rapidly aggregating.6 Similarly, we suggest that the folding and the stability of the UHM domain of MAN1 depend upon binding to a yet unknown biological partner.

The consequence of the unusual behavior of the predicted UHM domain is that most winged helix peaks can be easily identified in the HSQC spectrum of the entire carboxyl-terminal region of MAN1. Essentially no chemical shift perturbations were found between the spectrum of the winged helix domain alone and the spectrum of the winged helix domain within the entire carboxyl-terminal region. This suggests that there is no extensive contact between the winged helix and the predicted UHM domains of MAN1.

The Carboxyl-terminal Nucleoplasmic Region of MAN1 Binds to Both DNA and R-Smads—We carried out gel retardation assays using a 211-base pair linear DNA fragment and increasing quantities of either MAN1CA or MAN1CB. Clearly, both domains bind to DNA. The recognition helix H3 of the winged helix domain is critical for this binding, as it is in other published winged helix domain-DNA complexes. The apparent affinity for DNA is only 3-fold higher for MAN1CB as compared with MAN1CA, showing that the contribution of the poorly folded MAN1CB region to DNA binding is not essential.

MAN1CB also shows a significant affinity for the MH2 domain of R-Smads (8, 9). MAN1CA, which spans from amino acids 655 to 775 of MAN1 or the predicted UHM domain alone (amino acids 776-910 of MAN1) are not sufficient for R-Smad binding. However, the region from amino acid 730 to amino acid 910 of MAN1, comprising the beta2- and beta3-strands of the winged helix domain, the linker between this domain and the putative UHM domain, and the UHM domain itself, binds to Smad2 and Smad3 (9). Interestingly, it has been shown that several homeodomain and winged helix transcription factors recruit activated Smad2 to distinct promoter elements through an interaction between the MH2 domain of Smad2 and a common Smad binding motif (41). This motif is located after the DNA binding domain of these transcription factors and comprises the following sequence consensus, PPNKT/SI/VX3hX4-h, where h is a hydrophobic residue (42). In the case of MAN1, an analogous MH2 binding motif is found within the linker, between Pro-777 and Leu-785, 2 residues after MAN1CA and a few residues before the first beta-strand of the putative UHM domain. This motif PPNSLTX2L could participate to the MAN1-Smad2 interaction. Such a hypothesis is consistent with the reported unfolded structure of several free MH2 binding ligands. More generally, as both the two last beta-strands of the winged helix domain or the linker region and the putative UHM domain are necessary and sufficient for R-Smad binding and as these regions are essentially distinct from those suggested as critical for DNA binding, i.e. the recognition helix and the tip of the beta-hairpin (Fig. 6B), we propose that MAN1CB can bind simultaneously to DNA and the MH2 domain of R-Smads.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In the nucleus, Smads bind to transcriptional coactivators and promoter regions and play a role in the transcription of numerous genes regulated by transforming growth factor-beta superfamily members (43). The MH1 domain of Smads interacts with DNA. However, the affinity of Smads for DNA is relatively low, and Smads require other sequence-specific binding factors to bind efficiently to the promoters of certain responsive genes (44). The MH2 domain of Smads interacts with several such factors, which include a plethora of non-homologous proteins including FoxH1, Mixer, TGIF, CBP, AML1, Ski, and SIP1 (43). MAN1CA adopts a three-dimensional structural motif found in several of these proteins and binds to DNA with an apparent affinity of 10-7 M through its helix H3. This same helix mediates interaction with DNA in classical DNA binding winged helix domains. Furthermore, it was recently proposed that several Smad-associated proteins share a common MH2 binding motif (41). This motif is partially found in MAN1 25 amino acid residues after the carboxyl terminus of the winged helix domain at the amino terminus of the putative UHM domain. If MAN1 uses this motif to interact with R-Smads, then it can be predicted that the highly homologous human LEM2 protein, which shares the LEM domain, the two transmembrane segments, and the winged helix domain with MAN1 but lacks the linker and putative UHM regions, does not act as an antagonist of the Smad-mediated signaling pathways activated by bone morphogenetic protein, transforming growth factor-beta, or activin. Gotzmann and co-workers (30) recently reported that, consistently, they have not been able to detect antagonism of R-Smad-mediated signaling activity by LEM2. As LEM2 exhibits a highly positively charged helix H3 completely similar to MAN1 helix H3 in its winged helix domain, it could potentially regulate MAN1 DNA binding by interacting with the same specific DNA sequences. Finally, MAN1CB binds to other transcriptional regulators such as GCL and Btf (17). A competition between the different transcription regulators associated to MAN1 might also play a role in the complex regulation of the transcription of genes potentially targeted by the winged helix domain of MAN1.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2CH0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The chemical shift assignments reported here have been deposited in the BioMagRes-Bank Database under accession number 6919.

* 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 Supported by Grants 8699, 9513, and 11591 from Association Française contre les Myopathies. Back

2 Present address: United Biomedical, Inc., 25 Davids Dr., Hauppauge, NY 11788. Back

3 Supported by Grant MDA3711 from the Muscular Dystrophy Association. Back

4 To whom correspondence should be addressed. Tel.: 33-1-69-08-30-26; Fax: 33-1-69-08-90-71; E-mail: szinn{at}cea.fr.

5 The abbreviations used are: UHM, U2AF homology motif; r.m.s.d., root mean square deviation; TSP, 3-(trimethylsilyl)[2,2,3,3-2H4] propionate. Back

6 K. Frénal, personal communication. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Roger Genet, Marie Courcon, Mireille Moutiez, and Cedric Masson for their help during protein purification. We thank Philippe Savarin and Flavio Toma, who kindly lent us their 600-MHz spectrometer. The 900-MHz spectra were recorded at the SON NMR Large Scale Facility in Utrecht, which is funded by the Access to Research Infrastructures program of the European Union.



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 DISCUSSION
 CONCLUSIONS
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