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

Interactions between RA and PrTX-I atoms in the PrTX-I/RA complex.

<p>The surface charge distribution for the PrTX-I/RA crystallographic model and specific interactions between RA with some PrTX-I atoms are shown. Only the interactions with interatomic distances shorten than 3.7 Å are represented between chain A and the RA molecule (black dashes). To represent the interactions between RA and the residue Pro123 of chain B a larger distance cut-off was considered (blue dashes). Residues whose contacts with RA are established through water molecules are not shown. The electron density map for RA molecule was calculated with coefficients 2|F_obs|-|F_calc| contoured at 1.0 standard deviation.</p

Comparison of the PrtX-I/RA complex with two other Lys49-PLA₂s complexed to fatty acids.

<p>(a) Cartoon representation for the PrTX-I/RA complex. (b) PrTX-I/RA complex displayed in the same orientation shown in (a) shows RA occluding the entrance of the chain A hydrophobic channel. A surface representation is used for protomer A and cartoon representation with a semi-tranparent surface is employed for protomer B. The hydrophobic channel of protomer A is shown in yellow and the C-terminus of protomer B (residues 119–125) is represented in blue. (c) Superposition of PrTX-I/RA complex with PrTX-II/fatty acid (pink) and MjTX-II/stearic acid (light green) complexes (only one protomer of the homodimeric toxins is represented). (d) Surface view of the PrTX-I/RA complex shows that the hydrophobic channel of monomer A is in close contact with the C-terminal region of monomer B. The RA molecule is shown in sticks in all panels.</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

Effect of RA upon the muscle damage index induced by PrTX-I in mouse diaphragm preparations.

<p>The ordinate represents the % of damaged fibers relative to normal fibers and the abscissa indicates the experimental groups. The bars are expressed as mean ± S.E.</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 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

Effects of PrTX-I alone and PrTX-I pre-incubated with RA on indirectly evoked twitches in mouse phrenic-diaphragm preparations.

<p>The ordinate represents the percentage amplitude of twitches relative to the initial amplitude. The abscissa indicates the time after the addition of toxin to the organ bath. The points are the mean ± S.E. * indicates the point at which differences between PrTX-I and the control become significant.</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

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