Conformational heterogeneity of karyopherin beta2 is segmental - PubMed (original) (raw)

Conformational heterogeneity of karyopherin beta2 is segmental

Ahmet E Cansizoglu et al. Structure. 2007 Nov.

Abstract

Karyopherinbeta2 (Kap beta2) or transportin imports numerous RNA binding proteins into the nucleus. Kap beta2 binds substrates in the cytoplasm and targets them through the nuclear pore complex, where RanGTP dissociates them in the nucleus. Here we report the 3.0 A crystal structure of unliganded Kap beta2, which consists of a superhelix of 20 HEAT repeats. Together with previously reported structures of NLS and Ran complexes, this structure provides understanding of conformational heterogeneity that accompanies ligand binding. The Kap beta2 superhelix is divided into three major segments. Two of them (HEAT repeats 9-13 and 14-18), which constitute the substrate binding site, are rigid elements that rotate relative to each other about a flexible hinge. The third (HEAT repeats 1-8), which constitutes the Ran binding site, exhibits conformational changes throughout its length. An analogous segmental architecture is also observed in Importin beta, suggesting that it is functionally significant and may be conserved in other import karyopherins.

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Figures

Figure 1. Ribbon Diagrams of Unliganded and Substrate- and Ran-Bound Kapβ2s

β helices are represented as cylinders and structurally disordered loops as dashed lines. Unliganded Kapβ2 is in blue, Kapβ2 bound to substrate is in pink, Kapβ2 bound to Ran is in red, and H8 loops in all three structures are in yellow. Substrate hnRNP A1-NLS is in green and Ran is drawn as a surface representation in gray.

Figure 2. The Four Molecules of Unliganded Kapβ2 in the Asymmetric Unit Show Conformational Heterogeneity

(A) Four NCS-related Kapβ2s (chains A–D) are superimposed at H8–H13. The chains (each in a different shade of blue) are drawn as spheres at the geometric center of each HEAT repeat. Maximum deviations between geometric centers at the termini are shown. (B) Results of TLSMD analysis for partitioning of the four Kapβ2 chains into two to four TLS groups (each in a different color).

Figure 3. Hinge Motion in the C-Terminal Arch of Kapβ2

Stereo diagrams of the C-terminal arch (H9–H20) of chains A–D of unliganded Kapβ2 and substrate- and Ran-bound Kapβ2s superimposed at H9–H13 and drawn as spheres at the geometric center of each HEAT repeat. The hinge axes that rotate unliganded Kapβ2 with respect to substrate- and Ran-bound Kapβ2s are in pink and red, respectively.

Figure 4. Conformational Change in the Kapβ2 N-Terminal Arch

(A) Ribbon diagram of the N-terminal arches of chains A (blue) and C (light blue) of unliganded Kapβ2, superimposed at H9–H13. (B) Same as (A), except that chain A of unliganded Kapβ2 is superimposed on substrate-bound Kapβ2 (pink). (C) Same as (A), except that chain A of Kapβ2 is superimposed on Ran-bound Kapβ2 (red). Ran is shown as a surface representation in gray and the H8 loop of the Ran complex is in yellow.

Figure 5. The H8 Loop of Kapβ2 Is Mostly Disordered

The 2Fo – Fc map (stereo diagram, 1.0σ, blue mesh) of unliganded Kapβ2 at the H8 loop (yellow) and C-terminal arch (blue) shows that most of the loop is disordered and not bound in the C-terminal arch. H8 loop (yellow) residues 311–319 are shown connecting to H8A and residues 370–375 to H8B. No continuous density is observed beyond residue K319 until residue I370, indicating that the rest of the H8 loop is structurally disordered and no additional density is observed bound to the concave surface of the C-terminal arch. A zoomed-in view of the ordered portions of the H8 loop is shown in Figure S5.

Figure 6. Hinge Motion in Impβ and Kap95p Structures

(A) IBB-bound Impβ (brown) and SREBP2-bound Impβ (purple) are superimposed at HEAT repeats 5–10, and shown as spheres at the geometric center of each HEAT repeat. The hinge axis that rotates H1–H12 with respect to H14–H19 is shown in green. (B) RanGTP-bound Kap95p (light green) and Nup1p-bound Kap95p (orange) are superimposed at HEAT repeats 6–13, and shown as spheres at the geometric centers of their HEAT repeats. The hinge axis that rotates H1–H14 with respect to H15–H19 is shown in green.

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Conformational heterogeneity of karyopherin beta2 is segmental - PubMed (original) (raw)