Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA

Abstract

The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.

Fig. 1. Analytical ultracentrifugation of LAP2^1–168. Sedimentation equilibrium profiles, plotted in terms of ln(A₂₉₀) versus the square of the radius (r²), for intact LAP2^1–168 (open circles) and thombin-cleaved LAP2^1–168 (filled circles). In the case of thrombin-cleaved LAP2^1–168 the N-terminal His-tag and the polypeptide chain between Arg86 and Ser87 are completely clipped as judged by SDS–PAGE. The sample comprises 10 mg/ml LAP2^1–168 in 50 mM potassium phosphate pH 7.2 and the data were collected at 20°C and 16 000 r.p.m. The molecular weight for intact LAP2^1–168 is 20 700 ± 700, indicating that LAP2^1–168 exists as a single monomeric species in solution. The average molecular weight of thrombin-cleaved LAP2^1–168 is 9040 ± 200. These data indicate that LAP2^1–168 includes two independently folded domains that do not associate with each other in solution, consistent with the NMR data.

Fig. 2. Best-fit superpositions of the final 20 simulated annealing structures of (A) the LAP2-N domain and (B) the LAP2-C domain. The backbone is shown in red and ordered side chains in blue.

Fig. 3. (A) Ribbon diagrams of the LAP2-N domain (‘LEM-like’), the LAP2-C domain (LEM-domain), the C-terminal domain of T4 endonuclease VII and the N-terminal domain of the transcription termination factor rho. (B) Structure-based sequence alignment of the four domains. Residues in helices or helical turns are colored red. The coordinates of T4 endonuclease VII and rho were taken from Allison et al. (1998; PDB accession code 1A62) and Raaijmakers et al. (1999; PDB accession code 1EN7), respectively.

Fig. 4. ¹H-¹⁵N correlation spectra illustrating the interactions between LAP2^1–168, DNA and BAF. (A) Comparison of LAP2 in the absence (red) and presence of 1.5 equivalents (blue) of a duplex DNA dodecamer. Only cross-peaks of the LAP2-N domain shift upon DNA binding, while those of the LAP2-C domain remain unchanged, indicating that duplex DNA interacts exclusively with the LAP2-N (LEM-like) domain. (B) Comparison of the spectrum of LAP2^1–168 (selected region) in the absence (red) and presence (blue) of one equivalent (in terms of monomer) of BAF. Only the cross-peaks in the LAP2-C domain change significantly upon binding to BAF, while the cross-peaks of the LAP2-N domain remain unchanged. Thus, BAF only interacts with the LAP2-C domain (LEM-domain). (C) Comparison of LAP2^1–168 in the absence (red) and presence (blue) of the BAF–DNA nucleoprotein complex. All resolved peaks in the complex are labeled and arise from the LAP2-N domain. All peaks originating from the LAP2-C domain disappear upon binding the BAF–DNA nucleoprotein complex. These data indicate that only the LAP2-C domain interacts with the BAF–DNA nucleoprotein complex.

Fig. 5. Interaction surfaces on (A) LAP2-N for DNA, (B) LAP2-C for BAF and (C) BAF for LAP2-C derived from chemical shift mapping. The left side of the figure shows the domains as molecular surface representations with residues that either shift or broaden significantly upon binding colored in green (hydrophobic), cyan (hydrophilic), blue (positively charged) and red (negatively charged). For LAP2-N (A), Arg33 and Lys34 are displayed in magenta since they are clearly located in the DNA binding site but their backbone amide resonances were too broad to be observed in the ¹H-¹⁵N correlation spectra either in the free state or bound to DNA. For BAF (C), residues known to interact with DNA from single site mutational analysis are also displayed in magenta (Umland et al., 2000). To the right of the figure are ribbon diagrams in the same orientations as the molecular surfaces; best-fits of LAP2-N (red) and LAP2-C (blue) are displayed in (A) and (B); and the two subunits of BAF are shown in red and blue in (C). The views shown in (A) and (B) differ by an ∼90° rotation. The structures in (A) and (B) are displayed on the same scale, while that in (C) is on a smaller scale. Coordinates for BAF are from Cai et al. (1998; PDB accession code 1QCK).