Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex - PubMed (original) (raw)
Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex
M Peyro et al. Sci Rep. 2015.
Abstract
The nuclear pore complex (NPC) is the portal for bidirectional transportation of cargos between the nucleus and the cytoplasm. While most of the structural elements of the NPC, i.e. nucleoporins (Nups), are well characterized, the exact transport mechanism is still under much debate. Many of the functional Nups are rich in phenylalanine-glycine (FG) repeats and are believed to play the key role in nucleocytoplasmic transport. We present a bioinformatics study conducted on more than a thousand FG Nups across 252 species. Our results reveal the regulatory role of polar residues and specific sequences of charged residues, named 'like charge regions' (LCRs), in the formation of the FG network at the center of the NPC. Positively charged LCRs prepare the environment for negatively charged cargo complexes and regulate the size of the FG network. The low number density of charged residues in these regions prevents FG domains from forming a relaxed coil structure. Our results highlight the significant role of polar interactions in FG network formation at the center of the NPC and demonstrate that the specific localization of LCRs, FG motifs, charged, and polar residues regulate the formation of the FG network at the center of the NPC.
Conflict of interest statement
B.L. Lee’s work has been funded based upon work supported by National Science Foundation under Award No. 1132670. The rest of the authors declare no competing financial interests.
Figures
Figure 1. Comparison of FG Nups and DisProt proteins in terms of their charge content and distribution.
Top Row: Probability density distribution (a) and box plot (b) of mean absolute net charge, defined as absolute net charge divided by the length of the sequence. FG Nups exhibit lower absolute net charge compared to the DisProt database–please refer to “Methods” for the definition of database of FG Nups and Disprot. Interestingly, DisProt proteins show a much wider range of mean absolute net charge compared to FG Nups, implying that FG Nups share the same charge content (number density and distribution) characteristics across species. Bottom Row: Probability density distribution (c) and box plot (d) of number density of charged residues, defined as the number of charged residues divided by the length of the entire sequence. Sequences of FG Nups feature lower density of charged residues compared to DisProt. The two comparisons imply that charged residues are not only responsible for the disorderedness of FG Nups, but are also carefully distributed to give specific functionalities to FG Nups. Note that the area under each curve equals one, which explains why the values on vertical axes could exceed unity.
Figure 2. Distribution of charged residues along the sequences of yeast FG Nups.
Positive charges are shown in blue while negative charges are shown in red. The far right side of the figure represents the NPC wall. FG Nups are drawn in their extended form. It is clearly observable that not all domains of FG Nups are rich in charged residues, which implicitly proposes specific functionalities for different regions of each FG Nup (also discussed in and39). Yeast is chosen as the example to represent the distribution of charged residues in the sequence of FG Nups since it is the most well studied species.
Figure 3. Amino acid abundance of FG Nups and DisProt proteins.
Amino acids are sorted according to their disorder-promoting property, with Tryptophan as the most order-promoting amino acid and Proline as the most disorder-promoting one. Both groups show low abundance of hydrophobic residues (Ile, Leu, Val, Trp, Tyr, Phe) compared to structured proteins. However, FG Nups have a lower content (number density) of hydrophobic residues compared to those in DisProt proteins, except for Phenylalanine. Furthermore, FG Nups demonstrate higher abundance of polar residues and lower abundance of charged residues compared to DisProt proteins. Abundance is defined as the number of residue normalized by the number of residues in the entire database.
Figure 4. Comparison of central channel and periphery FG Nups in terms of number of charged residues embedded in LCR.
Data points are jittered to enable the reader to observe all of the data points. FG Nups fall into two distinct groups, with the central channel group possessing LCRs of lower than eight and the periphery group containing LCRs of larger than seven residues. The analysis is conducted on FG Nups from yeast and vertebrates with known location, i.e. central channel or periphery. Analyzed FG Nups are: [Central Channel] Nsp1 (blue), Nup49 (black), Nup57 (red), Nup145N (green), Nup62 (purple), Nup54 (yellow), Nup45 (gray), Nup58 (brown), [Cytoplasmic and Nuclear peripheries] Nup1 (orange), Nup2 (violet), Nup159 (cyan), Nup42 (blue), Nup100 (black), Nup116 (red), Nup98 (green), Nup358 (purple), Nup214 (yellow), Nup153 (gray), Nup50 (brown). Location information is obtained from.
Figure 5. Distribution of FG Nups in length-content space of LCRs.
Positive LCRs are shown in red while negative LCRs are shown in blue. Charge content here refers to the charge number density defined as number of charged residue normalized by the length of LCR. Negative LCRs are short and contain high charge density, while positive LCRs show very low charge density and longer sequences. In addition, positive LCRs have a wide range of lengths across different species.
Figure 6. Average polar residue content versus charged residue content in FG Nups and Disprot proteins.
For FG Nups, only FG linkers with the length of 10 to 30 residues are taken into account. For Disprot proteins, random sequences of 10–30 residues long are selected from Disprot proteins. For the sake of simplicity, these sequences from both groups are called linkers. Each point indicates the average number of polar residues among all linkers falling within that specific range of charged residue densities. Data points with at least 50 samples are shown (FG linkers with charged residue number densities of higher than 0.7 are excluded). Pie charts show average charged residue number density (red), average polar number density (dark brown), and average hydrophobic residue number density (cream) for the corresponding data point. Average number density of polar residues in FG Nups displays a sharp decreasing trend with respect to an increase in the number density of charged residues as compared to Disprot proteins, implying that FG Nups are more likely to have polar residues as the number of charged residues decreases. Disprot proteins are compared to FG Nups as a control group to show the significance of the observed evolutionarily conserved trend.
Figure 7. Overlap between charged and polar clusters.
The relatively low overlap indicates that charge-rich regions, termed stalk domains, have a low number density of polar residues. The overlap percentage calculation method is explained in the “Methods”, clustering method section. The same analysis was repeated on Disprot proteins as control group (Supplementary Figure S4). Relatively high overlap of polar and charged residues in Disprot shows the significance of the trend observed here.
Figure 8. Distribution of polar clusters along the sequences of FG Nups.
Right) A schematic showing half of the NPC from side-view. FG Nups are divided into three zones with zone1 representing the N-terminus and zone3 representing the C-terminus, which is mostly the structured region of the pore. Left). A heatmap showing abundance of polar clusters in the corresponding region. Brighter areas show higher content of polar clusters. FG Nups can be categorized into three groups. FG Nups of the first group are very rich in polar clusters in their first zone, while FG Nups of the second and third group have more polar clusters in their second and third zones, respectively.
Figure 9. Overlap between FG and polar clusters in three different categories of FG Nups.
Boxplots of the overlap among all FG Nups (red), FG Nups with more than half of their polar clusters located within the first zone (green), termed Group A, and FG Nups with more than half of their polar clusters located within second and third zones (blue), termed Group B are shown. Higher overlap of FG and polar clusters in group A implies a functional significance of overlapped FG-polar clusters at the tip of FG Nups.
Figure 10. Schematic demonstrating cooperation of positive LCRs, FG motifs, and polar residues to form the FG network.
Red spheres (top left box) represent positive charges embedded in positive LCRs, purple spheres (middle left box) represent FG motifs, and green spheres (bottom left box) represent polar residues within the central transporter. The highlighted FG Nups domain in the three left boxes represent N-terminal domain of NSP1, i.e. spheres represent location of charged (red) and polar (green) residues and FG motifs (purple). Due to the presence of positive LCRs, the central transporter is positively charged, facilitating transport of negatively charged NTRs. The dynamics of the central network of FG motifs is also regulated via charge repulsion in LCRs. In addition, low charge content of positive LCRs assists these domains of FG Nups to form a collapsed coil conformation. Furthermore, high densities of polar residues among FG repeats promote the hydrophobic interactions and meshwork formation. On the other hand, highly charged stalk domains of FG Nups, which are depleted of polar residues, place the central transporter in the center of the NPC by forming a relaxed coil conformation.
References
- Jamali T., Jamali Y., Mehrbod M. & Mofrad M. R. K. Nuclear Pore Complex. Biochemistry and Biophysics of Nucleocytoplasmic Transport in Health and Disease. International Review of Cell and Molecular Biology 287, (Elsevier Inc., 2011). -PubMed
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