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Originally published In Press as doi:10.1074/jbc.M700576200 on March 13, 2007

J. Biol. Chem., Vol. 282, Issue 19, 14525-14535, May 11, 2007
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Solution NMR Structure of the Barrier-to-Autointegration Factor-Emerin Complex*

Mengli Cai{ddagger}, Ying Huang§, Jeong-Yong Suh{ddagger}, John M. Louis{ddagger}, Rodolfo Ghirlando§, Robert Craigie§, and G. Marius Clore{ddagger}1

From the Laboratories of {ddagger}Chemical Physics and §Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0520

Received for publication, January 19, 2007 , and in revised form, February 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The barrier-to-autointegration factor BAF binds to the LEM domain (EmLEM) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF2 dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF2 and EmLEM. Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of EmLEM bound per BAF2 dimer. The equilibrium dissociation constant (Kd) for the interaction of the BAF2 dimer and EmLEM, determined by isothermal titration calorimetry, is 0.59 ± 0.03 µM. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF2 dimer in the complex yields a dissociation rate constant of 78 ± 2s-1. The solution NMR structure of the BAF2-EmLEM complex reveals that the LEM and DNA binding sites on BAF2 are non-overlapping and that both subunits of the BAF2 dimer contribute approximately equally to the EmLEM binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF2 dimer and EmLEM at the nuclear envelope is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The barrier to autointegration factor (BAF)2 (1) and the inner nuclear envelope LEM-domain protein emerin (2) are highly conserved cellular proteins throughout the metazoan kingdom that play an important role in nuclear architecture (3). BAF is an all-helical obligate dimer (4) that possesses two symmetry related DNA binding sites that permit BAF to bridge DNA chains and thereby compact DNA (5). Emerin is a member of the LEM (LAP2, Emerin, MAN1) family of nuclear proteins, and its loss is associated with the X-linked recessive form of Emery-Dreifuss muscular dystrophy (6). Emerin is a multidomain protein comprising an N-terminal globular LEM domain (EmLEM) of ~50 residues (7), followed by two polyserine segments separated by a hydrophobic nuclear localization signal, and a C-terminal transmembrane region. EmLEM comprises three helices (8) and is very similar in structure to the LEM domain of the related nuclear envelope protein LAP2 (9). BAF binds to EmLEM (as well as to the LEM domain of LAP2; Ref. 9) and is required for assembly of emerin at the nuclear envelope (10). BAF prevents autointegration of Moloney murine leukemia virus pre-integration complexes in vitro (1), and BAF and emerin have been reported to promote engagement of the HIV-1 pre-integration complex with chromatin prior to integration (11). To further our understanding of the interaction between BAF and the LEM domain of emerin we have characterized the stoichiometry of the complex by NMR, light scattering, and analytical ultracentrifugation; determined the equilibrium constant by isothermal titration calorimetry (ITC) and the dissociation rate constant by z-exchange spectroscopy; and solved the three-dimensional structure of the complex in solution by multidimensional heteronuclear NMR spectroscopy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Expression and Purification—The LEM domain (residues 1-47) of human emerin (7), EmLEM, was subcloned into a modified pET-32a vector (12) to form a thioredoxin fusion protein with a His6 tag and expressed in Escherichia coli strain BL21(DE3) (Novagen, La Jolla, CA). The construct was verified by DNA sequencing. E. coli transformed with the EmLEM vector was grown on either Luria Bertini or minimal medium (with 15NH4Cl and 13C6-glucose as the nitrogen and carbon sources, respectively), induced with 1 mM isopropyl D-thiogalactopyranoside at A600 ~ 0.8, and harvested by centrifugation 3 h following induction. After harvesting, the cell pellet was resuspended in 50 ml (per liter of culture) of 50 mM Tris, pH 7.4, 100 mM dine-Sepharose column (1 ml; Amersham Biosciences), followed by the addition of 1 mM phenylmethylsulfonyl fluoride. The cleaved His6-thioredoxin was removed by loading the digested proteins over a HisTrap HP column. EmLEM was further purified by a Sephadex-75 gel filtration column (Amersham Biosciences) equilibrated with 50 mM potassium phosphate, pH 6.5, and 0.01% (w/v) sodium azide. This buffer was used for NMR studies on free EmLEM.

Human BAF was expressed and purified as described (4). The following isotopically labeled samples were prepared: U-15N/13C-labeled, 10% 13C-labeled and unlabeled (natural isotopic abundance) EmLEM;U-15N/13C-labeled, U-15N/13C/2H/[methyl-1H]Val/Leu/Ile-labeled, 10% 13C-labeled and unlabeled BAF2 dimer. NMR samples of the BAF2-EMLEM complex were prepared in 50 mM potassium phosphate, pH 6.5, 200 mM NaCl, and 95% H2O, 5% D2O.

Light Scattering—Static light scattering data were obtained using an analytical Superdex-75 column (1.0 x 30 cm; GE Healthcare) with in-line multiangle light scattering (DAWN EOS, Wyatt Technology, Inc., Santa Barbara, CA) and refractive index detectors (OPTILAB DSP, Wyatt Technology Inc.). 145 µg of BAF2 dimer mixed with or without EmLEM in 125 µl of 50 mM potassium phosphate, pH 6.5, 200 mM NaCl was applied to the pre-equilibrated Superdex-75 column (1 x 30 cm; GE Healthcare) at a flow rate of 0.5 ml/min at room temperature and eluted in the same buffer.

Analytical Ultracentrifugation—Protein stocks purified in 50 mM potassium phosphate, pH 6.5, 200 mM NaCl, and 5 mM 2-mercaptoethanol were used to prepare the samples for sedimentation equilibrium experiments. Samples of the purified complex (eluted in a single peak by gel filtration chromatography) were studied at a loading concentration of 12 µM. Different stoichiometric BAF2 dimer to EmLEM mixtures were prepared at 3:1, 2:1, 1:1, 1:2, and 1:3 ratios and nominal BAF2 concentrations of 20 µM. The 2:1, 1:1, and 1:2 mixtures were also studied at nominal BAF2 concentrations of 13 µM. All samples were kept at 4 °C and loaded into pre-chilled cells.

Sedimentation equilibrium experiments were conducted at 4 °C on a Beckman Optima XL-A analytical ultracentrifuge. Samples of the complex and various BAF2/EmLEM mixtures were studied at rotor speeds of 16,000, 20,000, 24,000, and 28,000 rpm. Data were acquired using 6-hole cells as an average of 4 absorbance measurements at 280 nm and a radial spacing of 0.001 cm. Sedimentation equilibrium was achieved within 48 h. Data collected at different speeds and different loading concentrations were analyzed globally in terms of various species analysis models using SEDPHAT 4.1b (13) to obtain the buoyant molecular mass M(1 - v{rho}). A solution density {rho} of 1.01310 cm3/g was measured at 20 °C on a Mettler-Toledo DE51 density meter and corrected to a value of 1.0149 cm3/g at 4 °C, which is the value used experimentally. Partial specific volumes (v) for BAF and EmLEM (at 4 °C) were calculated based on the amino acid composition using SEDNTERP: the values are 0.7287 and 0.7184 cm3/g, respectively.

Isothermal Titration Calorimetry—ITC was performed using a high-precision VP-ITC calorimetry system (Microcal Inc.). BAF2 dimer and EmLEM were dialyzed against degassed 25 mM Tris-HCl buffer, pH 6.5, and 0.2 M NaCl prior to the experiment. BAF2 dimer (31 µM) in the calorimetric cell at 30 °C was titrated with EmLEM (at a concentration of 854 µM in the injection syringe). Analysis of the data were performed using the Origin software provided with the instrument.

NMR Spectroscopy—Spectra were recorded at 30 °C on Bruker DMX500, DRX600, DRX750, and DRX800 spectrometers. Spectra were processed using the program NMRPipe (15), and analyzed using the programs PIPP, CAPP, and STAPP (16). Sequential assignment of 1H, 15N, and 13C resonances was achieved by means of through-bond heteronuclear scalar correlations along the protein backbone and side chains (17, 18) using three-dimensional HNCOCACB, HNCACB, (H)C(CO)NH TOCSY, H(CCO)NH-TOCSY, and CCH-COSY experiments. Interproton distance restraints were derived from three-dimensional 15N- and 13C-separated NOE experiments. Stereospecific assignments of valine and leucine methyl groups were obtained from a 1H-13C HSQC correlation spectrum recorded on 10% 13C-labeled protein (19). Side chain rotamers were derived from 3JNC'(aromatic, methyl, and methylene) and 3JCC (aromatic, methyl, and methylene) scalar couplings measured by quantitative J correlation spectroscopy (20), in combination with data from a short mixing time (40 ms) three-dimensional 13C-separated NOE spectrum recorded in H2O (21). Intermolecular interproton distance restraints were derived from three-dimensional 12C-filtered/13C-separated NOE experiments recorded on complexes comprising either U-15N/13C or U-15N/13C/2H/[methyl-1H]Val/Leu/Ile BAF2 dimer complexed to unlabeled EmLEM, or U-15N/13C-labeled EmLEM complexed to unlabeled BAF2 dimer. Residual dipolar couplings (RDCs) were measured by taking the difference in J couplings between aligned (~15 and ~11 mg/ml phage pf1 (22) for free EmLEM and the BAF2-EMLEM complex, respectively) and isotropic media using well established procedures (23). For free EmLEM, 1DNH, 1DNC', and 2DHNC' RDCs were obtained. For the BAF-EmLEM complex DaNH RDCs were measured on complexes of 15N/13C-labeled BAF2 dimer and unlabeled EmLEM, and 15N/13C-labeled EmLEM and unlabeled BAF2 dimer (note only 1DNH RDCs are required for the structure determination of the complex because the backbones of the two proteins are treated as rigid bodies, see below; Ref. 24). The magnitudes of the axial (D NHa) and rhombic ({eta}) components of the alignment tensor for free EmLEM were obtained from a histogram of the distribution of the normalized RDCs (25). For the complex, D NHa and {eta} were obtained by singular value decomposition using the coordinates of the free proteins (23).

Z-exchange spectroscopy was carried out using the pulse sequence described previously (26, 27) using U-15N/13C/2H/[methyl-1H]Val/Leu/Ile-labeled BAF2 in the presence of 2, 3, and 4 eq of unlabeled EmLEM. The auto- and exchange-peak intensities as a function of mixing time were fitted by numerically integrating the appropriate McConnell (28) differential equations and optimizing the unknown parameters (dissociation rate constant, spin-lattice relaxation rate, and scale factors) using the program FACSIMILE (29), as described previously (27, 30).

Structure Calculations—Interproton distance restraints were derived from the NOE spectra and classified into generous approximate distance ranges corresponding to strong, medium, weak, and very weak NOE cross-peak intensities (21). Nonstereospecifically assigned methyl, methylene, and aromatic protons and ambiguous intermolecular NOEs were represented by a ({sum}r-6)-1/6 sum (31). {varphi}/{psi} torsion angle restraints for free EmLEM were derived from backbone (N, C', C{alpha}, Cbeta, H{alpha}) chemical shifts using the program TALOS (32). Side chain {chi} torsion angle restraints were derived from 3J heteronuclear couplings and short mixing time NOE experiments using standard procedures (21). The minimum range for the torsion angle restraints was ±20°.

All structure calculations were carried out using Xplor-NIH (33, 34) and the IVM (35) module for torsion angle and rigid body dynamics. The structure of the free EmLEM domain was calculated by simulated annealing in torsion angle space (35). The structure determination of the BAF2-EmLEM complex was carried out using conjoined rigid body/torsion angle dynamics (24, 35). The target function for simulated annealing comprises: square-well potentials for interproton distance and torsion angle restraints (36), harmonic potentials for 13C{alpha}/13Cbeta chemical shift restraints (37), RDC restraints (38), and covalent geometry; and a quartic van der Waals repulsion potential (39), a multidimensional torsion angle data base potential of mean force (40), a backbone hydrogen bonding data base potential of mean force (41), and a radius of gyration term (42) to represent the non-bonded contacts. The radius of gyration term represents a weak overall packing potential and the target value is given by 2.2N0.38, where N is the overall number of residues (42).

Structures were displayed using the VMD-XPLOR software (43). Reweighted atomic probability density maps used to represent the conformational space sampled by the interfacial side chains within the complex were calculated and displayed as described previously (44).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Structure Determination of the Free EmLEM Domain—The structure of the free EmLEM domain was determined on the basis of 820 experimental NMR restraints, including 110 backbone RDCs. A summary of the structural statistics is provided in Table 1, a stereoview of the superposition of the ensemble of 180 simulated annealing structures is shown in Fig. 1A, and a ribbon diagram is provided in Fig. 1B. The structure comprises a 3-10 helix (residues 2-6) and two {alpha}-helices (residues 9-19 and 28-46) oriented at an angle of 43° to one another. The structure of EmLEM is very similar to our previously published structure of the BAF binding LEM domain of LAP2 (9) with a C{alpha} atomic r.m.s. difference of 1.3 Å for 44 atoms (residues 2-46 of EmLEM and 111-154 of LAP2; percentage sequence identity of 36%). The C{alpha} atomic r.m.s. difference between the current EmLEM structure and the NMR structure previously published by Wolff et al. (8) is 2.4 Å for residues 2-46 and 1.5 Å for residues 3-44. Although the fold and topology of the two EmLEM structures are obviously the same, there are clearly differences in detail, which are significant when one wants to use the coordinates of the free EmLEM domain to solve the structure of the BAF2-EMLem complex using conjoined rigid body/torsion angle dynamics. In this regard, we note that the agreement of the EmLEM coordinates of Wolff et al. (8) with the 1DNH RDCs measured for EmLEM both free and bound to the BAF2 dimer is rather poor with RDC R-factors (46) of 49 and 61%, respectively, determined by singular value decomposition. In contrast, the present structure of the EmLEM domains agrees extremely well with the 1DNH RDCs measured on the BAF2-EmLEM complex with an RDC R-factor of 14.8%. The latter value is comparable with the value one would expect for a 1.5-2-Å resolution crystal structure (23, 47, 48) (Note that the overall orientation of the alignment tensors of free EmLEM and the BAF2-EMLEM complex differ by 128°; hence the RDCs measured for EmLEM in the BAF2-EmLEM complex provide a good cross-validation measure of the quality of the coordinates of free EmLEM.)


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  		TABLE 1
Structural statistics for free EmLEM

<SA> are the final 180 simulated annealing structures. (SA)r is the restrained regularized mean structure derived from the mean coordinates obtained by averaging the coordinates of the 180 simulated annealing structures best-fitted to each other. The number of terms for the various experimental restraints is given in parentheses. None of the structures exhibit interproton distance violations >0.3 Å or torsion angle violations >5°.

 
Stoichiometry of the BAF2-EMLEM Complex by NMR—The BAF2-EmLEM complex is in slow exchange on the chemical shift scale and portions of the 1H-15N HSQC spectra recorded as a function of various ratios of EmLEM to BAF2 dimer are shown in Fig. 2A. The binding of EmLEM to the BAF2 dimer disrupts the symmetry of the dimer such that the chemical shifts of many equivalent residues of the two subunits (about 55% of the 1H-15N cross-peaks for BAF) are no longer identical in the complex. Under the conditions of the NMR experiment (concentration of BAF2 dimer of ~130 µM), binding of EmLEM is stoichiometric with one molecule of EmLEM bound per BAF2 dimer (Fig. 2B). Increasing the ratio of EmLEM to BAF2 dimer above 1:1 results in no change in the intensity of the bound BAF2 cross-peaks (Fig. 2, A and B).


Figure 1
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  		FIGURE 1.
Structure of the EmLEM domain. A, superposition of 180 simulated annealing structures with the backbone (N, C{alpha}, C') atoms in red, and selected side chains in blue. B, ribbon diagram of the EmLEM domain with the 3-10 helix and two {alpha}-helices indicated in different shades of purple.

 
Because a single molecule of EmLEM binds to the BAF2 dimer, the chemical environments of the two subunits of BAF2 are necessarily no longer identical. The backbone of the two subunits of BAF2, however, remains identical within coordinate errors as judged from RDC measurements (i.e. the values of the 1DNH RDCs for the two subunits of BAF2 are identical in the complex). It should also be noted that if two molecules of EmLEM bound the BAF2 dimer symmetrically, the chemical environment and hence the chemical shifts of the two subunits of BAF2 would be identical in the complex.

Stoichiometry of the BAF2-EMLEM Complex by Light Scattering—The calculated molecular mass of the BAF2 dimer and the EMLEM domains are 20,116 and 5,572 Da, respectively. The BAF2 dimer elutes as a single peak with a molecular mass of 21.3 ± 0.2 kDa determined from light scattering and the refractive index data (Fig. 3A). A 1:1 mixture of BAF2 dimer and EmLEM results in a shift of the BAF2 peak to a lower retention volume with a molecular mass of 25.9 ± 0.2 kDa (Fig. 3B). Increasing the ratio of EmLEM to BAF2 does not change the position of the latter peak and the molecular mass obtained at a ratio of BAF2 dimer to EmLEM of 1:2, 1:3, and 1:4 is 24.7 ± 0.2, 24.8 ± 0.2, and 27.4 ± 0.2 kDa, respectively. The peak eluting at ~15.4 ml with a molecular mass of 6.48 ± 0.02, 6.45 ± 0.01, and 6.76 ± 0.01 kDa shown in Fig. 3, C-E, respectively, corresponds to free EmLEM. These results clearly indicate that the BAF2-EmLEM complex comprises one BAF2 dimer and one molecule of EmLEM. The concentration of complex upon elution is ~12 µM. The observation that the position of the peak corresponding to the complex does not change upon increasing concentration of EmLEM indicates that the equilibrium dissociation constant for the complex is ≤1 µM.

Stoichiometry of the BAF2-EMLEM Complex by Analytical Ultracentrifugation—Sedimentation equilibrium experiments on the BAF2-EmLEM complex purified by size-exclusion gel filtration chromatography were carried out at rotor speeds of 16,000 to 28,000 rpm and analyzed in terms of a single ideal solute. Excellent data fits were obtained (Fig. 4) returning a molecular mass of 26.8 ± 0.4 kDa. Based on the amino acid sequence and solution density, the BAF2 dimer and EmLEM monomer have calculated molecular masses of 20,116 and 5,572 Da, respectively, indicating that the complex has a 2:1 BAF:EmLEM stoichiometry (n = 1.04 ± 0.02): that is one molecule of EmLEM for two subunits of BAF monomer. To confirm the stoichiometry of the complex, sedimentation equilibrium experiments were carried out on a 1:1 loading mixture of BAF2 dimer and EmLEM at concentrations of 13 and 20 µM BAF2 dimer. A global data analysis in terms of a single ideal solute returned excellent fits with an experimental molecular mass of 25.8 ± 0.3 kDa (Fig. 4), confirming the formation of a 2:1 BAF: EmLEM complex (n = 1.00 ± 0.01).

To show that these species only form a 2:1 BAF:EmLEM complex, various BAF2 dimer and EmLEM mixtures were studied. In the presence of excess BAF2, namely the 3:1 and 2:1 BAF2:EmLEM loading ratios, free BAF2 dimer (molecular mass of 20,116 Da) and the 2:1 BAF:EmLEM complex (molecular mass of 25,688 Da) are the only species expected. As the molecular masses are too similar to distinguish by sedimentation equilibrium, a mixture of these two species represents a so-called paucidisperse system and an analysis in terms of a single ideal solute should return a weight average molecular mass. The 3:1 BAF2 dimer to EmLEM loading mixture returns a weight average molecular mass of 22.2 ± 0.2 kDa with excellent data fits (data not shown), consistent with the formation of the 2:1 BAF-EmLEM complex. Such a loading mixture is expected to return a weight average molecular mass of 22,147 Da if the 2:1 BAF-EmLEM complex were formed. If the BAF-EmLEM complex had a 2:2 stoichiometry (i.e. one EmLEM molecule per BAF subunit), a weight average molecular mass of 24,334 Da would be expected. Data collected for the 2:1 BAF2/EmLEM loading mixture were also consistent with a 2:1 BAF-EmLEM complex stoichiometry, within the error of the method.


Figure 2
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  		FIGURE 2.
NMR titration of EmLEM and BAF2. A, portions of the 1H-15N HSQC spectra of 15N/13C-labeled BAF2 dimer (~130 µM) free and in the presence of 0.5, 1, and 4 eq of EmLEM. Cross-peaks of free BAF2 are labeled with the superscript F; cross-peaks for the two subunits of bound BAF2 that are no longer chemically equivalent because of breaking of symmetry from the presence of EmLEM are labeled with the superscripts B and B'. B, 1H-15N cross-peak intensity for the N{epsilon}-H{epsilon} group of Trp62 (closed and open circles correspond to the peaks labeled with the superscripts B and B', respectively, in the lower panels of A.

 
Sedimentation equilibrium experiments carried out in the presence of excess EmLEM, namely 1:2 and 1:3 BAF2 dimer to EmLEM mixtures, could not be modeled adequately in terms of a single ideal solute. Accordingly, data were analyzed in terms of two ideal solutes, of which one represents the free EmLEM domain. Fixing the molecular mass of the smaller species to 5,572 Da, a 1:2 BAF2 dimer to EmLEM mixture returns a molecular mass of 27.0 ± 0.95 kDa for the second species with excellent data fits (data not shown). These data are consistent with the sole formation of a 2:1 BAF-EmLEM complex (n = 1.05 ± 0.04). A 1:3 BAF2 dimer to EmLEM mixture containing 18.4 µM BAF2 dimer and 55.2 µM EmLEM returns a molecular mass of 29.2 ± 1.5 kDa (n = 1.14 ± 0.06) (data not shown). Thus the stoichiometry of the BAF2-EmLEM complex, comprising one molecule of EmLEM bound per BAF2 dimer, is unambiguously confirmed by three independent techniques covering a range of concentrations and molar ratios.

Equilibrium and Kinetic Characteristics of the BAF2-EMLEM Complex—ITC was used to determine the equilibrium and thermo-dynamic properties of the interaction of EmLEM with the BAF2 dimer. The experimental ITC binding curve obtained upon addition of EmLEM toa31 µM solution of BAF2 dimer is shown in Fig. 5A. A best-fit to the experimental ITC data with a stoichiometry of one EmLEM molecule bound per BAF2 dimer yields an equilibrium dissociation constant (Kd) of 0.59 ± 0.03 µM, corresponding to a binding free energy ({Delta}G) of -8.63 ± 0.03 kcal mol-1, and an enthalpy ({Delta}H) of -5.70 ± 0.05 kcal mol-1. Thus, the interaction of EmLEM with BAF2 is entropically favored with {Delta}S = 9.7 cal mol-1 K-1, where {Delta}S = ({Delta}H-{Delta}G)/T, and T is the temperature in Kelvin. The increase in entropy upon complex formation arises from the hydrophobic effect (49) as a consequence of the displacement of ordered water at the binding interfaces of the two proteins, and suggests that the interaction between EmLEM and BAF2 is predominantly stabilized by hydrophobic interactions.

The kinetics of the interaction of unlabeled EmLEM and U-15N/13C/2H/[methyl-1H]Val/Leu/Ile BAF2 were studied by z-exchange spectroscopy (26, 27), which revealed the presence of chemical exchange cross-peaks between the two sets of shifts for the BAF2 dimer in the BAF2-EmLEM complex (Fig. 5B). This arises from the fact that EmLEM can bind to the BAF2 dimer in two chemically equivalent ways related by a 180° rotation about the C2 symmetry axis of the BAF2 dimer (Fig. 6). Thus cross-peaks corresponding to the two magnetically inequivalent subunits of BAF2 in the complex are simply interchanged in the two bound states. The exchange process occurs via dissociation followed by reassociation (and note both orientations are equally probable because the two possible complexes are chemically equivalent) (Fig. 5C). The McConnell (28) differential equations describing the evolution of magnetization as a function of mixing time for the scheme shown in Fig. 5C are as follows,

Formula(Eq.1)

Formula(Eq.2)

Formula(Eq.3)


Figure 3
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  		FIGURE 3.
Size exclusion chromatography and multiangle light scattering on the BAF2-EmLEM complex. Elution profiles from an analytical S75 column (1 x 30 cm) monitored by refractive index of BAF2 dimer alone (A) (145 µg in a 125-µl injection volume) and in the presence of 1 (B) (40 µg), 2 (C) (80 µg), 3 (D) (120 µg), and 4 (E) (160 µg) eq of EmLEM. Assuming a ~5-fold dilution of the sample in the column, the concentration of BAF2 and BAF2-EmLEM complex is ~12 µM upon elution. The molecular masses obtained from the light scattering and refractive index measurements correspond to a BAF2 dimer, BAF2 dimer-EmLEM complex, and free EmLEM shown in blue, red, and green closed circles, respectively.

 


Figure 4
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  		FIGURE 4.
Analytical ultracentrifugation on the BAF2-EmLEM complex. Sedimentation equilibrium profiles obtained for the purified BAF2-EmLEM complex (left) and a 1:1 BAF2/EmLEM mixture (right) shown in terms of A280 versus the radius r for data collected at a loading concentration of 12 µM complex (left) and 13 µM BAF2 (right). Data were collected at 4 °C and 16,000 (green), 20,000 (blue), 24,000 (yellow), and 28,000 (red) rpm. For clarity, alternate data points have been omitted. In both cases data were analyzed in terms of a single ideal solute to return a molecular mass consistent with that of a 2:1 BAF-EmLEM complex (i.e. one molecule of EmLEM per BAF2 dimer). Best single ideal solute fits are shown as black lines through the experimental points. The corresponding distributions of the residuals are shown in the plots above. The data set shown for the 1:1 mixture was analyzed globally along with a similar sample having 20 µM BAF2 dimer.

 
where MF is the magnetization of free BAF2, and MB and MB' are the magnetizations for the two species of BAF2 in the complex related by the 180° rotation of bound EmLEM; kon and koff are the association and dissociation rate constants, respectively;{rho}F and {rho}B are the spin-lattice relaxation rates for BAF2 in the free and bound states, respectively; [EmLEM]free is the concentration of free EmLEM; and kon[EmLEM]free is a pseudo-first order rate constant because the concentration of free EmLEM is not perturbed during the experiment. The intensity of a given auto-peak and its associated exchange-peaks as a function of mixing time are obtained by numerical integration of Equations 1-3 with the magnetization of the species corresponding to the auto-peak set to 1 and the magnetization of the species corresponding to the exchange-peaks set to zero.

Z-exchange experiments were carried out at three different concentrations of free EmLEM, 0.39, 0.68, and 0.89 mM. The evolution of the intensities of the normalized auto- and exchange-peaks as a function of mixing time was found to be concentration independent (Fig. 5D). This is as expected because kon·[EmLEM]free >> koff, so that the apparent rate of interconversion between the magnetizations MB and MB' is koff/2 in each direction. Note also that the calculated maximum magnetization of the exchange-peak for MF is less than 10-3, and hence no exchange-peak corresponding to free BAF2 is observed. Simultaneous best-fitting of the time courses of the intensities of the auto- and exchange-peaks (Fig. 5D) yields a value of koff = 78 ± 2s-1. Given the value of Kd determined by ITC, the association rate constant (kon) is calculated to be ~1.3 x 108M-1s-1, typical of a diffusion-controlled protein-protein association reaction (50).


Figure 5
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  		FIGURE 5.
Thermodynamics and kinetics of the BAF2-EmLEM complex. A, ITC titration of BAF2 with EmLEM. The titration (3 µl per injection of 854 µM EmLEM) was performed at 30 °C in a calorimetric cell (~1.8 ml) containing 31 µM BAF2 dimer in 25 mM Tris-HCl, pH 6.5, and 0.2 M NaCl. The experimental data are shown as solid circles. The best-fit curve to a one site binding equilibrium is shown as a solid line and yields a value of Kd = 0.59 ± 0.03 µM. B, because one molecule of EmLEM binds to the BAF2 dimer, the chemical environments of equivalent residues from the two subunits of BAF are no longer identical and display different chemical shifts, as illustrated for Gly47. Z-exchange spectroscopy reveals the presence of exchange cross-peaks (indicated by ex) between equivalent residues in addition to the auto-peaks (labeled as G47 and g47'). This arises from the fact that EmLEM can bind to the BAF2 dimer in two chemically equivalent ways related by a 180° rotation (see Fig. 6). C, kinetic scheme describing the magnetization transfer involving dissociation and reassociation of EmLEM to BAF2 in two chemically equivalent orientations. Cross-peaks corresponding to the two magnetically inequivalent subunits of BAF2 in the complex are simply interchanged in the two bound states. MF is the magnetization of free BAF2; MB and MB' are the magnetizations of the two bound states of BAF2 related by the 180° rotation of EmLEM; kon and koff are the association and dissociation rate constants, and [EmLEM]F is the concentration of free EmLEM; {rho}F and {rho}B are the spin-lattice relaxation rates for free and bound BAF2 and for simplicity are considered equal because {rho}F cannot be determined from the present data. D, time course of the normalized auto-(open circles) and exchange-(closed circles) peaks of Gly47 together with the best-fit curves (red and blue lines, respectively) obtained for the kinetic model shown in C. The experimental data are shown at three different concentrations of free EmLEM (0.39, 0.68, and 0.89 mM) with total concentrations of U-15N/13C/2H/[methyl-1H]Val/Leu/Ile-labeled of 0.42, 0.35, and 0.30 mM, respectively, and total concentration of unlabeled EmLEM of 0.81, 1.03, and 1.19 mM, respectively. E, selected strips from a three-dimensional 12C-filtered/13C-separated NOE spectrum illustrating intermolecular NOEs from 12C-attached protons of the BAF2 dimer (F1 dimension) to 13C-attached protons of EmLEM (F3 dimension). The spectrum was recorded in 95% H2O, 5% D2O. The cross-peaks involving equivalent residues in the two subunits of BAF2 are indicated by upper and lowercase one-letter codes.

 
It should be noted that the exchange process observed by z-exchange spectroscopy is a phenomenon that can only be observed by NMR and is of no functional significance because the two binding orientations are chemically equivalent and therefore functionally identical. (It is, of course, of biophysical significance because it enables one to determine the value of the dissociation rate constant.) The existence of the exchange process does, however, have implications for the NMR structure determination of the complex. In particular, all intermolecular NOEs must be treated as ambiguous ({sum}r-6)-1/6 sums (31) because no distinction can be made a priori as to which BAF subunit is involved in a given intermolecular NOE. This situation is exactly analogous to the situation that pertains to the {delta} and {epsilon} protons of Phe and Tyr residues undergoing 180° ring flips. It should also be noted that because of chemical exchange between the two binding orientations (related by a 180° rotation), an NOE cross-peak from a residue of EmLEM to a residue on one subunit of BAF will be transferred by chemical exchange to the corresponding residue on the other BAF subunit. This is clearly evident in some of the strips taken from a three-dimensional 12C-filtered/13C-separated NOE spectrum shown in Fig. 5E.


Figure 6
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  		FIGURE 6.
EmLEM binds to the BAF2 dimer in two chemically equivalent orientations related by a 180° rotation about the C2 axis of symmetry of the BAF2 dimer. The two subunits of the BAF2 dimer are shown in red and blue; and the two orientations of EmLEM are shown in green and purple. The structure shown represents the restrained regularized mean coordinates derived from an ensemble of 180 simulated annealing structures calculated using conjoined rigid body/torsion angle dynamics (see Fig. 7).

 
Structure Determination of the BAF2-EmLEM Complex—The structure of the BAF2-EmLEM complex was solved by conjoined rigid body/torsion angle dynamics (24, 35) on the basis of 308 experimental NMR restraints including 140 backbone 1DNH RDCs that yield precise and accurate orientational restraints related to the relative positions of BAF2 and EmLEM within the complex, and 31 intermolecular interproton distance restraints derived from three-dimensional 12C-filtered/13C-separated NOE spectroscopy that provide translational, as well as orientational, information. In these calculations the backbone and non-interfacial side chains for the two proteins are treated as rigid bodies, whereas the interfacial side chains are given full torsional degrees of freedom (24, 35): the backbone of the BAF2 dimer is held fixed, EmLEM is free to rotate and translate, and the single RDC alignment tensor is free to rotate. The starting coordinates employed for the complex comprise the published NMR structure of the BAF2 dimer (Protein Data Bank code 1QCK; Ref. 42) and the present NMR structure of the EMLem domain (the restrained regularized mean coordinates are used because these are the coordinates that are closest to the mean yet satisfy the experimental restraints as well as any of the individual simulated annealing structures; Ref. 51). Within the errors of the NMR coordinates there are no changes in backbone conformation upon complex formation: the 1DNH RDCs recorded on the complex agree with the coordinates of free BAF2 dimer and EmLEM with RDC R-factors (46) of 15.2 and 14.8%, respectively, which is comparable with values expected for 1.5-2-Å resolution crystal structures (47, 48). The RDC R-factors for BAF2 and EmLEM in the refined complex (that is using a single alignment tensor for the whole complex) are the same as those obtained by singular value decomposition fitting to the coordinates of the two proteins individually. A table of structural statistics for the BAF2-EmLEM complex is provided in Table 2. A superposition of the back-bone for an ensemble of 180 simulated annealing structures is shown in Fig. 7A, and reweighted atomic probability density maps for the interfacial side chains, derived from the ensemble, are shown in Fig. 7, B and C.


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  		TABLE 2
Structural statistics for the BAF2-EmLEM complex

The notation is the same as that in Table 1. The number of experimental restraints for the various terms is given in parentheses.

 


Figure 7
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  		FIGURE 7.
Stereoviews of the NMR structure of the BAF2-EmLEM complex. A, superposition of the backbone (N, C{alpha}, C') atoms) of 180 simulated annealing structures (EmLEM, green, and the two subunits of the BAF2 dimer shown in red and blue). Reweighted atomic probability density maps (drawn at a value of 25% maximum and calculated from the final 180 simulated annealing structures) for the interfacial side chains, illustrating the interactions between EmLEM (gray mesh) and the red (B) and blue (C) subunits of the BAF2 dimer (red meshes). The backbone (with the same color scheme as in A) is represented by flat ribbons. The side chains of the restrained regularized mean coordinates are color coded according to atom type (carbon, gray; oxygen, red; and nitrogen, blue).

 
The BAF-Emerin Interface—The interaction surface between the BAF2 dimer and EmLEM is formed by a convex protrusion on EmLEM comprising helix {alpha}1, the subsequent loop, and the N-terminal end of {alpha}2; and a deep concave cleft on the BAF2 dimer comprising the C-terminal end of {alpha}2, the subsequent hairpin turn and {alpha}3 of the red subunit of BAF, and the hairpin turn between {alpha}2 and {alpha}3, the C-terminal end of {alpha}3, and the central portion of {alpha}4 of the blue subunit of BAF (Fig. 8A). (For clarity we distinguish the two subunits of BAF by color coding.) There is no overlap between the single EmLEM binding site and the two symmetry related DNA binding sites on the BAF2 dimer. The latter comprises the N terminus of {alpha}1, the 3-10 helix/turn/{alpha}2 motif, and the N-terminal portion of {alpha}5. 969 Å2 of accessible surface area are buried at the interface of which 462 Å2 originates from BAF2 and 507 Å2 from EmLEM. The loop connecting helices {alpha}1 and {alpha}2ofEmLEM interact with the red subunit of BAF, whereas {alpha}1 and the following loop interact with the blue subunit. The binding surfaces on both BAF2 and EmLEM consist of a central hydrophobic portion surrounded by a rim of polar and charged residues (Fig. 8B), typical of many protein-protein complexes (47). The key hydrophobic interactions involve Val51, Leu52, Leu58, val51, phe39, and gly38 of BAF (where lowercase letters indicate residues from the blue subunit) and Leu23, Gly24, Phe25, and Val26 of EmLEM (italics denote residues of EmLEM). The preponderance of hydrophobic interactions at the interface and the displacement of ordered water from these hydrophobic surfaces upon binding accounts for the positive entropic change upon complex formation observed by ITC. Key electrostatic interactions occur between Arg37, Glu61, and Asp65 of the red subunit of BAF and Asp9, Lys38, and Lys36, respectively, of EmLEM (Fig. 7B) and between glu36 of the blue subunit of BAF and Arg17 of EmLEM, and possibly the hydroxyl groups of Thr10 and Thr13 via water-mediated interactions as well (Fig. 6C). Additional electrostatic interactions include possible water bridged contacts between Gln48 of the red subunit of BAF and the hydroxyl groups of Ser29 and Thr30 of EmLEM (Fig. 7B). Trp62 of the red subunit of BAF is principally involved in hydro-phobic contacts with Thr30 and Leu33 (Fig. 7B), and trp62 of the blue subunit with Val27 (Fig. 7C). The observed interactions between BAF and EmLEM are fully consistent with mutagenesis data that showed that the G24A/P25A/V26A/V27A, T30A/ R31A, and Y34A/E35A/K36A/K37A mutations significantly reduce binding of emerin to BAF (52). (Note that a fourth emerin mutation that disrupts BAF binding, E11A/L12A (52), does not involve the interaction surface but is predicted to destabilize the EmLEM core through the introduction of a cavity as a consequence of the replacement of a leucine by the much smaller alanine side chain.)


Figure 8
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  		FIGURE 8.
The BAF2-EmLEM interface. A, ribbon diagram of the BAF2-EmLEM complex (color coded as in Fig. 7A) also illustrating the position of the two DNA duplexes observed in the crystal structure of the BAF2-DNA2 complex (5). B, surface representations illustrating the binding surfaces involved in the BAF2-EmLEM complex. The binding surface on BAF2 is shown on the left panel and on EmLEM on the right panel. The binding surfaces are color coded with hydrophobic residues in green, polar residues in light blue, positively charged residues in dark blue, and negatively charged residues in red. The relevant portions of the interacting partner are shown as gold tubes. The surface of the non-interacting residues of the BAF2 dimer is shown in dark gray for the red subunit and light gray for the blue subunit (as depicted in A). Residues of the blue subunit of BAF2 are labeled in lowercase, and residues of EmLEM in italics. The view in the right-hand panel is related to that in the left-hand panel by a 180° rotation about an axis parallel to the printed lines on the page.

 
Modulation of the Interaction of BAF with LEM Domain Proteins—The structure of the BAF2-EmLEM complex reported here, together with the structure of BAF2 in complex with DNA (5), places constraints on how the interaction of BAF with LEM domain proteins is regulated. BAF and LEM domain proteins function as part of large nucleoprotein networks; attempts to fish out interacting partners of BAF and LEM domain proteins by biochemical techniques yields numerous proteins, most of which presumably interact indirectly.3 LAP2 was first identified as a BAF-interacting protein in a yeast two-hybrid screen, and deletion analysis mapped a region encompassing the LEM domain to be sufficient for this interaction (53). The structure of the BAF2-EmLEM complex establishes the basis of this interaction. In in vitro binding studies, the LAP2 constant region has a higher affinity for BAF bound to DNA than for BAF alone and this was taken as evidence for a conformational change in BAF upon DNA binding (54). It is now clear that no conformational changes in BAF occur upon binding either DNA or the LEM domain. Alternative explanations for the higher affinity of the LAP2 constant domain for BAF bound to DNA include the possible interaction of regions outside of the LEM domain with DNA or stabilization of the complex through binding of multiple units of BAF to DNA. Modulation of the BAF-LEM interaction by regions outside the LEM domain is also suggested by the different affinities of various LAP2 isoforms for BAF (54). In addition, studies of the behavior of fluorescently labeled BAF and emerin in cells also suggest modulation of the BAF-LEM interaction (55). A direct interaction between BAF and emerin at the nuclear envelope was demonstrated by FRET analysis. However, fluorescence recovery after photobleaching experiments showed that whereas BAF was highly mobile at the nuclear envelope, emerin was much less mobile. On the basis of these results a "touch and go" model was proposed in which BAF binds emerin frequently but transiently during interphase. This association of BAF and emerin agrees nicely with the transient interaction (koff ~ 78 s-1) we observe by NMR between BAF and the LEM domain of emerin. In contrast, BAF associates much more stably with LEM domain proteins at the "core" region of telophase chromosomes (55). This stable interaction cannot be accounted for by the interaction of BAF with the LEM domain alone, which is transient, and additional protein factors are likely involved.

Concluding Remarks—The structures of the BAF2-EmLEM and BAF2-DNA2 (5) complexes provides a structural basis for how BAF both bridges DNA and binds nuclear membrane proteins that contain the LEM domain. The BAF dimer is required for DNA bridging, but binding of the BAF dimer to a single LEM domain ensures that each BAF dimer interacts with only a single LEM-domain protein and prevents assembly of mixed complexes with multiple nuclear envelope proteins.


   FOOTNOTES
 
The atomic coordinates and experimental NMR restraints (accession codes 2ODC for free EmLEM and 2ODG for the BAF2-EmLEM complex) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by grants from the Intramural Research Program of the NIDDK, National Institutes of Health and by the AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C. and R. C.). 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 To whom correspondence should be addressed: Bldg. 5, NIDKK, National Institutes of Health, Bethesda, MD 20892-0520. Tel.: 301-496-0788; Fax: 301-496-0825; E-mail: mariusc{at}intra.niddk.nih.gov.

2 The abbreviations used are: BAF, barrier-to-autointegration factor; NOE, nuclear Overhauser effect; RDC, residual dipolar coupling; HSQC, hetero-nuclear single quantum coherence; ITC, isothermal titration calorimetry; r.m.s., root mean square. Back

3 R. Craigie, unpublished data. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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