Crystallographic data collection and refinement statistics of ΔIBB-h-importin-α1.

<p>^a<i>R</i>_merge = ΣΣ_<i>i</i> ||<i>I</i>(<i>h</i>)—<i>I</i>(<i>h</i>)_<i>i</i> | / ΣΣ_<i>i</i><i>I</i>(<i>h</i>), where <i>I</i>(<i>h</i>) is the mean intensity after rejections.</p><p>^b<i>R</i>_work = Σ| F_p—F_pc /Σ |F_p|.</p><p>^c<i>R</i>_free, the same as <i>R</i>_work but calculated on 4.93% of data excluded from refinement.</p><p>^d Clashscore, calculated by MolProbity.</p><p>Values in parentheses are for highest resolution shells.</p><p>Crystallographic data collection and refinement statistics of ΔIBB-h-importin-α1.</p

Crystal Structure of Human Importin-α1 (Rch1), Revealing a Potential Autoinhibition Mode Involving Homodimerization

<div><p>In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB)-truncated human importin-α1 (ΔIBB-h-importin-α1) at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.</p></div

Crystal structure of homodimeric ΔIBB-h-importin-α1.

<p>(A) The closed homodimer structure of ΔIBB-h-importin-α1. One of the protomers is shown as a surface drawing. Both of the P1′-binding pockets are depicted in close-up views. The K108 inserts in the P1′-binding pocket, making hydrogen bonds with D325, T328, and Q369 of another protomer, and Wat (a water molecule). The pseudo 2-fold axis is drawn in the close-up view for the center of the dimer. (B) The P1′ binding pocket with 2fo-fc electron density maps (&gt;1.0σ). Residues involved in the P1′-binding pocket are V321, T322, D325, T328, N361, I362, and Q369. One water molecule is depicted as Wat. The K108 of another protomer is inserted into the P1′-binding pocket, making hydrogen bonds depicted with dotted blue lines. (C) A ribbon drawing of the homodimeric ΔIBB-importin-α1 in cyan and gray. Residues of each protomer are in blue and orange. Major and minor NLS binding sites are indicated. Residues involved in the major NLS binding sites are W142, N146, W184, N188, W231, N235, W273, and Y277 in bold characters. The residues in the minor NLS biding sites are D325, T328, W357, N361, Q369, W399, and N403. Each of the K108 makes hydrogen bonds with D325, T328, Q369, and one water molecule in the minor NLS binding site of another protomer. Because the major and minor NLS binding sites are extensively buried in the dimerization interface as shown in the drawing, NLS signals are inaccessible to the sites. All molecular pictures were prepared with PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115995#pone.0115995.ref040" target="_blank">40</a>].</p

Autoinhibition states of h-importin-α1 by IBB binding and homodimerization.

<p>(A) A conventional scheme of autoinhibition by self-binding of IBB domain. The IBB domain bound in the NLS binding sites autoinhibits NLS binding. In addition, the current work reveals the IBB domain also prevents it from homodimerization. In turn, the bound IBB domain dissociates from the NLS binding sites by binding of NLS peptides and/or importin-β. (B) A potential homodimer autoinhibition mode. The current study reveals that a novel autoinhibition state by homodimerization, which possibly corresponds to NLS binding regulation.</p

AUC-SV measurements.

<p>(A) c(s) distributions of ΔIBB-h-importin-α1, 247 μM (purple), 100 μM (blue), 14 μM (cyan), 11 μM (green), 7.3 μM (yellow) and 4.2 μM (orange). (B) Normalized c(s) distributions of ΔIBB-h-importin-α1. (C) KD value estimation by the fitting curve of the sw vs protein concentration. (D) 25 μM ΔIBB-h-importin-α1 + SV40 NLS of 0 μM (purple), 1 μM (blue), 10 μM (cyan) and 100 μM (green). (E) 25 μM ΔIBB-h-importin-α1 + nucleoplasmin NLS of 0 μM (purple), 10 μM (cyan), and 100 μM (green). (F) Normalized c(s) distributions of h-importin-α1, 61 μM (purple), 34 μM (blue), 8.6 μM (cyan), and 4.3 μM (green). (G) 10 μM h-importin-α1 + SV40 NLS of 0 μM (purple), 10 μM (blue), 100 μM (cyan). (H) 10 μM h-importin-α1 + nucleoplasmin NLS of 0 μM (purple), 10 μM (blue), 100 μM (cyan).</p

ITC fitting curves.

<p>(A) h-importin-α1 + SV40 NLS peptide. (B) ΔIBB-h-importin-α1 + SV40 NLS peptide. (C) ΔIBB-h-importin-α1 + nucleoplasmin NLS. (D) h-importin-α1 + nucleoplasmin.</p