Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal

<div><p><i>Neurospora crassa</i> is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that <i>N. crassa</i> Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.</p></div

Schematic diagram of the interactions between SV40NLS and NcImp<i>α</i> for major (A) and minor (B) binding sites.

<p>The SV40NLS peptide main and side-chains are drawn in magenta. NcImp<i>α</i> residues interacting with the peptide are indicated by yellow (side-chain) and orange (main-chain). Polar contacts are shown as dashed lines and arcs with radiating spokes indicate hydrophobic contacts.</p

Comparison among Imp<i>α</i> crystal structures from different organisms.

<p>The Imp<i>α</i> proteins were superimposed using the C<i>α</i> atoms of each protein (residues 83 -505) <b>(a)</b> NcImp<i>α</i>, MmImp<i>α</i> (PDB ID: 3UL1) and HsImp<i>α</i>1 (PDB ID: 4WV6) are represented in black, red and orange, respectively. <b>(b)</b> NcImp<i>α</i>, OsImp<i>α</i> (PDB ID: 4BQK), ScImp<i>α</i> (PDB ID: 1BK5) and HsImp<i>α</i>5 (PDB ID: 2JDQ) are represented in black, blue, green and pink, respectively.</p

Binding to specific pockets of Imp<i>α</i>/NLS structures from different organisms.

<p>* Binding to specific binding pockets of Imp<i>α</i> based on structural data are shown in bold. The NLSs are aligned as observed to bind to the NLS-binding sites (</p><p></p><p></p><p></p><p><mi>P</mi><mn>1</mn><mo>′</mo></p><p></p><p></p><p></p> –<p></p><p></p><p></p><p><mi>P</mi><mn>4</mn><mo>′</mo></p><p></p><p></p><p></p>, minor binding site; <i>P</i>₁—<i>P</i>₆, major binding site, as defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref011" target="_blank">11</a>]). The suspension points corresponds to the extension of the NLS peptides that not bind into the linker region. NcImp<i>α</i>/SV40NLS; OsImp<i>α</i>/SV40NLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref016" target="_blank">16</a>]; MmImp<i>α</i>/SV40NLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref010" target="_blank">10</a>]; ScImp<i>α</i>/SV40NLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref009" target="_blank">9</a>];MmImp<i>α</i>/CN-SV40NLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref037" target="_blank">37</a>]; MmImp<i>α</i>/hPLSCR4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref038" target="_blank">38</a>]; MmImp<i>α</i>/TPX2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref039" target="_blank">39</a>]; MmImp<i>α</i>/Nucleoplasmin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref010" target="_blank">10</a>]; MmImp<i>α</i>/N1N2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref007" target="_blank">7</a>]; MmImp<i>α</i>/RB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref007" target="_blank">7</a>]; MmImp<i>α</i>/FEN1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref040" target="_blank">40</a>]; MmImp<i>α</i>/Bimax1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref041" target="_blank">41</a>]; MmImp<i>α</i>/Bimax2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref041" target="_blank">41</a>]; OsImp<i>α</i>/A89 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref016" target="_blank">16</a>]; OsImp<i>α</i>/B54 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref016" target="_blank">16</a>]; ScImp<i>α</i>/c-Myc [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref011" target="_blank">11</a>]; HsImp<i>α</i>5/Nup50 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref042" target="_blank">42</a>].<p></p><p>Binding to specific pockets of Imp<i>α</i>/NLS structures from different organisms.</p

Isothermal calorimetric data for SV40NLS peptide binding to NcImp<i>α</i>.

<p><b>(a)</b> Raw power output (<i>μ</i>cal/s) per unit time (min) of replicate titrations <b>(b)</b> Integrated data (kcal.mol⁻¹ of injectant versus molar ratio of SV40NLS to NcImp<i>α</i>). These data were obtained from the raw power output as the area underneath each peak, which is then corrected for baseline heat injections and SV40NLS dilution heat and mixing. The solid line represents the best fit of the data.</p

Isothermal titration calorimetry data for SV40NLS binding to NcImp<i>α</i>.

<p>Isothermal titration calorimetry data for SV40NLS binding to NcImp<i>α</i>.</p

X-ray data-collection and refinement statistics for NcImp<i>α</i>/SV40NLS structure.

<p>† </p><p></p><p></p><p></p><p><mi>R</mi></p><p><mi>m</mi><mi>e</mi><mi>r</mi><mi>g</mi><mi>e</mi></p><p></p><mo>=</mo><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo stretchy="false">(</mo><p><mo>∑</mo><mi>i</mi></p><mo stretchy="false">(</mo><mo stretchy="false">∣</mo><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi><mo>,</mo><mi>i</mi></p><p></p><mo>−</mo><mo><</mo><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo>></mo><mo stretchy="false">∣</mo><mo stretchy="false">)</mo><mo stretchy="false">)</mo><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi><mo>,</mo><mi>i</mi></p><p></p><mo><</mo><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo>></mo><p></p><p></p><p></p><p></p><p></p>, where <i>I</i>_<i>i</i> (hkl) is the intensity of an individual measurement of the reflection with Miller indices hkl and hI(hkl)i is the mean intensity of this reflection. Calculated for I > -3<i>σ</i> (I) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref021" target="_blank">21</a>].<p></p><p>‡ </p><p></p><p></p><p></p><p><mi>R</mi></p><p><mi>c</mi><mi>r</mi><mi>y</mi><mi>s</mi><mi>t</mi></p><p></p><mo>=</mo><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo stretchy="false">(</mo><mo stretchy="false">∣</mo><mo stretchy="false">∣</mo><mi>F</mi><mi>o</mi><mi>b</mi><p><mi>s</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo stretchy="false">∣</mo><mo>−</mo><mo stretchy="false">∣</mo><mi>F</mi><mi>c</mi><mi>a</mi><mi>l</mi><p><mi>c</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo stretchy="false">∣</mo><mo stretchy="false">)</mo><p></p><p><mo stretchy="false">∣</mo><mi>F</mi><mi>o</mi><mi>b</mi></p><p><mi>s</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><mo stretchy="false">∣</mo><p></p><p></p><p></p><p></p><p></p>, where ∣<i>Fobs</i>∣ and ∣<i>Fcalc</i>∣ are the observed and calculated structure-factor amplitudes, respectively.<p></p><p>§ <i>R</i>_<i>free</i> is equivalent to <i>R</i>_<i>cryst</i> but was calculated with reflections (5%) omitted from the refinement process. Calculated based on the Luzzati plot with the program SFCHECK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref031" target="_blank">31</a>].</p><p>† † Calculated with the program PROCHECK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128687#pone.0128687.ref031" target="_blank">31</a>].</p><p>^<i>a</i>Values in parentheses are for the highest-resolution shell.</p><p>X-ray data-collection and refinement statistics for NcImp<i>α</i>/SV40NLS structure.</p

Electron-density map (coefficients 2∣<i>F</i>_<i>obs</i>∣ − ∣<i>F</i>_<i>calc</i>∣) of the NcImp<i>α</i>/SV40NLS structure in the area corresponding to SV40NLS peptides (contoured at 2.0 s.d.) at major (A) and minor (B) binding sites.

<p>Electron-density map (coefficients 2∣<i>F</i>_<i>obs</i>∣ − ∣<i>F</i>_<i>calc</i>∣) of the NcImp<i>α</i>/SV40NLS structure in the area corresponding to SV40NLS peptides (contoured at 2.0 s.d.) at major (A) and minor (B) binding sites.</p

Comparison of SV40NLS peptides binding to the major (a) and minor (b) binding sites for Imp<i>α</i> structures from different organisms.

<p>SV40NLS peptides in complex with NcImp<i>α</i> (magenta), ScImp<i>α</i> (green), OsImp<i>α</i> (blue) and MmImp<i>α</i> (yellow) were superimposed using the C<i>α</i> atoms of the peptides. Positions binding corresponding to the major (P₁-P₅) and minor (P₁′-P₄′) sites are identified along the chains (binding pocket labels were based on peptide binding with NcImp<i>α</i>).</p