Self-Assembling Nano-Architectures Created from a Protein Nano-Building Block Using an Intermolecularly Folded Dimeric <i>de Novo</i> Protein

The design of novel proteins that self-assemble into supramolecular complexes is an important step in the development of synthetic biology and nanotechnology. Recently, we described the three-dimensional structure of WA20, a <i>de novo</i> protein that forms an intermolecularly folded dimeric 4-helix bundle (PDB code 3VJF). To harness the unusual intertwined structure of WA20 for the self-assembly of supramolecular nanostructures, we created a protein nanobuilding block (PN-Block), called WA20-foldon, by fusing the dimeric structure of WA20 to the trimeric foldon domain of fibritin from bacteriophage T4. The WA20-foldon fusion protein was expressed in the soluble fraction in Escherichia coli, purified, and shown to form several homooligomeric forms. The stable oligomeric forms were further purified and characterized by a range of biophysical techniques. Size exclusion chromatography, multiangle light scattering, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) analyses indicate that the small (S form), middle (M form), and large (L form) forms of the WA20-foldon oligomers exist as hexamer (6-mer), dodecamer (12-mer), and octadecamer (18-mer), respectively. These findings suggest that the oligomers in multiples of 6-mer are stably formed by fusing the interdigitated dimer of WA20 with the trimer of foldon domain. Pair-distance distribution functions obtained from the Fourier inversion of the SAXS data suggest that the S and M forms have barrel- and tetrahedron-like shapes, respectively. These results demonstrate that the <i>de novo</i> WA20-foldon is an effective building block for the creation of self-assembling artificial nanoarchitectures

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

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

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

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 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

Decreased Amyloidogenicity Caused by Mutational Modulation of Surface Properties of the Immunoglobulin Light Chain BRE Variable Domain

Amyloid formation by immunoglobulin light chain (LC) proteins is associated with amyloid light chain (AL) amyloidosis. Destabilization of the native state of the variable domain of the LC (V_L) is known to be one of the critical factors in promoting the formation of amyloid fibrils. However, determining the key residues involved in this destabilization remains challenging, because of the existence of a number of intrinsic sequence variations within V_L. In this study, we identified the key residues for destabilization of the native state of amyloidogenic V_L in the LC of BRE by analyzing the stability of chimeric mutants of BRE and REI V_L; the latter immunoglobulin is not associated with AL amyloidosis. The results suggest that the surface-exposed residues N45 and D50 are the key residues in the destabilization of the native state of BRE V_L. Point mutations at the corresponding residues in REI V_L (K45N, E50D, and K45N/E50D) destabilized the native state and increased amyloidogenicity. However, the reverse mutations in BRE V_L (N45K, D50E, and N45K/D50E) re-established the native state and decreased amyloidogenicity. Thus, analyses using chimeras and point mutants successfully elucidated the key residues involved in BRE V_L destabilization and increased amyloidogenic propensity. These results also suggest that the modulation of surface properties of wild-type V_L may improve their stability and prevent the formation of amyloid fibrils

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p