Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19 - PubMed (original) (raw)

Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19

Johanna Napetschnig et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 A resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The 6D7A loop of the Nup214 NTD is essential for Ddx19 binding. (A) Domain organization of Nup214 and Ddx19. For Ddx19, the N-terminal extension (NTE, orange) and the 2 RecA-like domains (domain 1 and 2, green and light orange) are indicated. For Nup214, the β-propeller domain (light blue) and its C-terminal extension (CTE, yellow), the coiled-coil domain (gray), and the C-terminal unstructured region containing numerous FG-repeats (white) are indicated. The Nup214 NTD is composed of the β-propeller domain followed by the CTE. The bars above the domain structures mark the fragments of the orthorhombic crystal form. (B) Gel filtration profiles of full-length wild-type Ddx19 incubated with the Nup214 NTD, Nup214 NTD 1–405, or Nup214 NTD Δ6D7A before injection.

Fig. 2.

Fig. 2.

Overview of the Nup214 NTD·Ddx19 NTD structure. Ribbon representation of the Nup214 NTD·Ddx19 NTD complex (Upper). A 90° rotated view is shown in Lower. For the Nup214 NTD, the β-propeller domain (blue), the 6D7A loop (magenta), the C-terminal extension (CTE; yellow), and the blade numbers are indicated. For the Ddx19 NTD, the N-terminal RecA-like domain (green) and the unique N-terminal extension (NTE; orange) is indicated. The ADP molecule bound to the Ddx19 NTD is shown in ball-and-stick representation.

Fig. 3.

Fig. 3.

Surface properties of the Nup214 NTD-Ddx19 NTD interaction. (A) Surface renditions of the NTDs of Nup214 and Ddx19 in an open book representation colored according to the participation of the various domains as in Fig. 2. Surfaces that mediate the association between the 2 proteins are indicated in green (Ddx19) and blue (Nup214). As a reference, a ribbon representation of the complex is shown in its original orientation. (B) Surface representation colored according to a multispecies sequence alignment (

Fig. S1

). The conservation at each position is mapped onto the surface and is shaded in a color gradient from light yellow (60% similarity) to dark red (100% identity). (C) Surface representation colored according to the electrostatic potential. The electrostatic potential is plotted onto the surface and colored in a gradient from red (−10 k_B_T/e) to blue (+10 k_B_T/e). The orientation of all surface representations is identical. Black lines indicate the interface borders.

Fig. 4.

Fig. 4.

The conserved arginine 259 of Ddx19 is a key residue for complex formation. (A) Details of the interaction between the NTDs of Nup214 and Ddx19. The ribbon representation is colored according to Fig. 2. The Inset illustrates the position of R259 and its interacting residues and is expanded on the right. (B) Multispecies sequence alignment of Ddx19 homologs. The red asterisk indicates the location of the invariant R259. The conserved sequence motifs II and III are highlighted in gray boxes. Invariant k_B_T residues outside of the conserved sequence motifs and R259 are illustrated in red. The residue numbering is relative to human Ddx19 and the secondary structure of Ddx19 is shown above the sequence alignment.

Fig. 5.

Fig. 5.

A 9-residue region in the 6D7A loop of the Nup214 NTD is essential for Ddx19 binding. Gel filtration profiles of full-length wild-type Ddx19 incubated with the deletion mutants Nup214 NTDΔ1, Δ2, or Δ3 before injection. Gel filtration profiles of Ddx19 are colored in gray, the Nup214 NTD variants in blue, and the elution profile resulting from incubating Ddx19 with Nup214 proteins in red. The red arrow indicates the expected elution volume of the Nup214·Ddx19 complex (see Fig. 1_B Top_).

Fig. 6.

Fig. 6.

Arginine 259 of Ddx19 is crucial for binding to Nup214 NTD. Gel filtration profiles of the Nup214 NTD (blue), the Ddx19 mutants R259A, R259Q, R259K and R259E (gray), and the elution profile resulting from incubation of Nup214 NTD with Ddx19 (red) before injection are indicated. The red arrow indicates the expected elution volume of the Nup214·Ddx19 complex (see Fig. 1_B Top_).

Fig. 7.

Fig. 7.

In vivo localization of Ddx19 and Ddx19 mutants. HeLa cells were transfected with Ddx19 and Ddx19 mutants containing a C-terminal HA-tag and analyzed with confocal microscopy. The monoclonal antibody mAb414 was used as a reference for nuclear envelope staining (red). The cellular localization of HA-tagged proteins was detected with an anti-HA antibody (green). The merged image reveals the colocalization of wild type Ddx19 and Ddx19 R259K with the nuclear envelope, while Ddx19 R259A displays no detectable nuclear envelope staining.

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