PDBsum entry 2odc

Membrane protein

PDB id: 2odc

Contents

Protein chain

47 a.a. *

* Residue conservation analysis

PDB id:

2odc

Name:

Membrane protein

Title:

Lem-domain of the nuclear envelope protein emerin

Structure:

Emerin. Chain: i. Fragment: residues 2-47. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Cellular_location: nucleus. Gene: emd, edmd, sta. Expressed in: escherichia coli. Expression_system_taxid: 562

NMR struc:

1 models

Authors:

G.M.Clore,M.Cai

Key ref:

M.Cai et al. (2007). Solution NMR structure of the barrier-to-autointegration factor-Emerin complex. J Biol Chem, 282, 14525-14535. PubMed id: 17355960 DOI: 10.1074/jbc.M700576200

Date:

22-Dec-06 Release date: 13-Mar-07

Protein chain

P50402  (EMD_HUMAN) -  Emerin from Homo sapiens

Seq: 254 a.a.

Struc: 47 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M700576200

J Biol Chem 282:14525-14535 (2007)

PubMed id: 17355960

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex.

M.Cai, Y.Huang, J.Y.Suh, J.M.Louis, R.Ghirlando, R.Craigie, G.M.Clore.

ABSTRACT

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) 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 BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.

Selected figure(s)

Figure 5.
FIGURE 5. Thermodynamics and kinetics of the BAF[2]-Em^LEM complex. A, ITC titration of BAF[2] with Em^LEM. The titration (3 µl per injection of 854 µM Em^LEM) was performed at 30 °C in a calorimetric cell ( 1.8 ml) containing 31 µM BAF[2] 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 K[d] = 0.59 ± 0.03 µM. B, because one molecule of Em^LEM binds to the BAF[2] 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 Gly^47. 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 Em^LEM can bind to the BAF[2] 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 Em^LEM to BAF[2] in two chemically equivalent orientations. Cross-peaks corresponding to the two magnetically inequivalent subunits of BAF[2] in the complex are simply interchanged in the two bound states. M[F] is the magnetization of free BAF[2]; M[B] and M[B]['] are the magnetizations of the two bound states of BAF[2] related by the 180° rotation of Em^LEM; k[on] and k[off] are the association and dissociation rate constants, and [Em^LEM][F] is the concentration of free Em^LEM; [F] and [B] are the spin-lattice relaxation rates for free and bound BAF[2] and for simplicity are considered equal because [F] cannot be determined from the present data. D, time course of the normalized auto-(open circles) and exchange-(closed circles) peaks of Gly^47 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 Em^LEM (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 Em^LEM 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 BAF[2] dimer (F[1]dimension) to ^13C-attached protons of Em^LEM (F[3] dimension). The spectrum was recorded in 95% H[2]O, 5% D[2]O. The cross-peaks involving equivalent residues in the two subunits of BAF[2] are indicated by upper and lowercase one-letter codes.

Figure 8.
FIGURE 8. The BAF[2]-Em^LEM interface. A, ribbon diagram of the BAF[2]-Em^LEM complex (color coded as in Fig. 7A) also illustrating the position of the two DNA duplexes observed in the crystal structure of the BAF[2]-DNA[2] complex (5). B, surface representations illustrating the binding surfaces involved in the BAF[2]-Em^LEM complex. The binding surface on BAF[2] is shown on the left panel and on Em^LEM 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 BAF[2] 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 BAF[2] are labeled in lowercase, and residues of Em^LEM 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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 14525-14535) copyright 2007.

Figures were selected by an automated process.