Nucleo cytoplasmic trafficking
2. Our Research Projectse. NPC Proteins, Nucleoporins and disease
Nucleoporins are essential for NPC formation and function. So, their improper localization and mutations could severely interfere with functionality and as a consequence with health. We are trying to understand both: How do nucleoporins fulfill their function with respect to interaction with transport receptors, what consequences might this have on the receptors and how mutations could interfere with the proper assembly and function of the NPCx resulting in diseases. Fig. 1 indicates the approximate location of three proteins we have investigated.
Fig.1. Schematic representation of the NPC. (A) Structural organization of the NPC and positioning of the nucleoporins, that have been worked on in the lab. (B) Electron tomography image of the human NPC (EMDBid: 2443; Bui et al., 2013) in surface representation (beige-brown) with schematic insertion of the individual nucleoporin families, color coded as presented in 1.a. Fig. 1. EMDB: Electron microscopy data base: http://www.emdatabank.org/.
NUP85 is an essential member of the Y-complex located on both sides of the nuclear membrane, forming the respective rings, whereas Nup 88 is located solely on the cytoplasmic side more peripheral to the membrane forming a complex with Nup214 a protein bearing FG repeats important for interaction to transport receptors and enabling selective transport.
A. Interaction with transport complexes
The importance of FG-repeats has already been mentioned in Fig 1. We try to understand the properties of FG-repeats by determining their interaction with transport receptors in order to explain how they might propagate facilitated diffusion through the NPCs central channel.
The structure of CRM1-RanGTP-SPN1 export complex with a fragment of the nucleoporin NUP214 encompassing FG-repeats revealed their mode of interaction (Fig. 2.)
Fig.2. Interaction pattern of MBP-Nup214 Fragment FG repeats to the CRM1-SPN1-RanGTP Complex. (A) Overall structure of the Nup214 interacting export complex. Three FG regions of Nup214 (red, orange, and yellow) bind to three distinct, mainly hydrophobic FG-binding patches on CRM1 (gray to white-gradient-colored surface from N to C terminus). RanGTP (green) is engulfed by the N-terminal region, the acidic loop, and C-terminal HEAT repeats of CRM1. SPN1 (blue) binds to the outer surface of CRM1 via two epitopes: the NES residues and the cap-binding domain. MBP, which was fused to the N terminus of Nup214 for crystallization, was omitted for clarity. It is located in front of SPN1, preceding the FG region 1 (indicated by a red asterisk; compare Figure S2A). (B–D) Detailed views of FG region 1 (red), FG region 2 (orange), and FG region 3 (yellow) of Nup214 bound to the respective FG-binding patches of CRM1. HEAT repeats are labeled and colored alternately in gray and white. Nup214 is shown in cartoon mode and as sticks. Phenylalanines of the FG-repeats are illustrated by transparent spheres and labeled.
The structure reveals eight binding sites for the Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Interestingly, Nup214 binds to both, N- and C-terminal regions of CRM1, thereby clamping the CRM1terminal regions in a closed conformation and stabilizing the export complex. In general, with respect to the binding mode of the phenylalanine side chains of Nup214 FG motifs, they neatly dock into hydrophobic surface pockets of CRM1, which are formed by hydrophobic side chains of amino acid residues of neighboring HEAT helices. Some of these hydrophobic surface pockets have also been identified in CRM1 structures lacking FG-binding partners, indicating their accessibility in solvent.
All of the Importin-ß superfamily share an overall superhelical structure owing to the tandem arrangement of a specific motif, the HEAT repeat. This structural organization leads to great intrinsic flexibility as demonstrated using various structures from the Protein Data Bank (Fig. 3.), which in turn is a prerequisite for the interaction with a variety of proteins and for its transport function. During the passage from the aqueous cytosol into the nucleus, the receptor passes the gated channel of the nuclear pore complex filled with a protein meshwork of unknown organization, which seems to be highly selective owing to the presence of FG-repeats, which are peptides with hydrophobic patches.
Fig.3. Importin-ß reveals high flexibility, enabling the binding of various proteins at different sites. Impß is depicted in rainbow coloring from the N-terminus (blue) to the C-terminus (red). Binding partners are depicted in violet. The labelling above indicates the organism from which the respective gene is derived (sc, Saccharomyces cerevisiae; mm, Mus musculus; cl, Canis lupus; hs, Homo sapiens); PDB codes are indicated. The extended triangle indicates the decrease in extension of the Impß structures presented.
In a more theoretical approach using two of our own Impß structures (Fig.4) and structural information available in the database we analyzed this intrinsic flexibility of Importin-ß by comparing free Importin-ß (Fig. 4) or different complexes from a single organism, crystallized in both, polar (salt) or apolar (PEG) buffer conditions. This allowed analysis of structural changes attributable only to the surrounding milieu and are not affected by bound interaction partners. The importin-ß structures obtained exhibit significant conformational changes and suggest an influence of the polarity of the environment, resulting in an extended conformation in the PEG condition.
Fig.4. Importin-ß adopts altering shape and elongation when crystallized in different conditions. Chaetomium thermophilum Impß was crystallized in an ammonium sulfate condition (light grey; PDB entry 4xri) and a PEG condition (dark grey; PDB entry 4xrk). The residues chosen for distance measurements are shown as spheres, the respective distances are indicated as lines (colored as the respective residues) and the distances are indicated in Angstrom beside/below the individual structures. The line at the bottom of the structure indicates the diameter in the N-terminal region (respective residues are shown in violet) and the top line indicates the diameter in the C-terminal region (orange). The green line at the side represents the pitch, i.e. the distance between two residues (green spheres) one helical turn apart, and the red line indicates the distance between two conserved, distant residues (red spheres). Note that HEAT repeat 1 could not be built in the structure obtained from the PEG condition.
B. NPC proteins: role in diseases
The effects of mutations in Nups with respect to diseases have become more and more eminent over the recent years and have already been described (Dickmanns et al 2015). The structural knowledge available on the individual Nups and their location and interaction partners in the overall structure of the NPC now allows to locate and understand their function on a structural level within the context of the NPC.
In collaboration with Labs in Bruxelles and Berlin we are analyzing the effect of mutations of Nups and the influences they might have on their individual structure as well as the effects on neighboring Nups using structures available and modelling tools.
Two examples, Nup 88 and Nup 85 are given below: Nup88 is part of a complex with Nup214, located on the cytoplasmic side of the NPC both of which seem to play a role a final binding sites for export complexes. mRNA export requires Nup88 whereas Nup214 interacts with CRM1 as shown above (2.d.).
Nup 85 is part of the y-complex which forms the inner and outer ring of the NPC on the nuclear membrane.
Biallelic mutations in the nucleoporin Nup88 cause lethal fetal akinesia deformation sequence (FADS). FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement.
Fig.5. Modelling of human NUP88. (A) The N-terminal domain of NUP88 reveals a seven-bladed ß-propeller with an N-terminal extension. The rainbow coloring indicates N-terminal residues in blue (NTD = N-terminal domain) and C-terminal residues of the propeller in red. Individual blades are indicated by numbers. The red arrow indicates the location of the p.D434Y point mutation (see below for details). (B) Overlay of the NUP88 model (red) with the Xray structures of Nup82 from baker’s yeast (PDBid: 3pbp, in gray) and C. thermophiles (PDBid: 5cww; in light yellow). Significant differences between species are in blade 4 (HTH-motif; yellow arrow) and 5 (extended loop; green arrow).(C) Composite model of the N- and C-terminal regions of NUP88. The presented model was generated using RaptorX with its standard settings and misses about 40 amino acid residues after the propeller region. Both, the propeller and CTD regions are colored in rainbow coloring). The individual mutations are indicated by their numbering and represented in sphere mode. (D) Magnification of the loop bearing the D434 mutation in NUP88 in stick mode. The coloring of the individual molecules is as described in (B).
Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH–SCKS) include a heterogeneous group of autosomal recessive inherited diseases. They are characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Nup85 is part of the socalled Y complex, an integral part of the cytoplasmic and nuclear rings. The structural analysis suggests that the identified NUP85 variants induce conformational changes that could have an effect on its interaction with other NUPs and thus on the overall NPC architecture (Fig. 6.).
Fig.6. Putative effect of identified variants on NUP85 and NPCs. NUP85 is an essential member of the NUP107–160 complex (Y-complex), representing the scaffold unit of the NPC. (B) Model NUP107–160 complex derived from PBDid: 5a9q with the individual components indicated. (C) Structure of human NUP85 based on PDBid 5a9q and an ITasser model (cyan lines) and the structure from M. thermophila (grey lines). Rainbow-coloring from N- (blue)
to C-terminus (red) within the trans blade in the N-terminal region. The remaining part is all helical with some helices indicated by numbers. The first helix (dark blue) marks the beginning of a U-shaped helical arrangement with helix 8 at the turning point, and then heading back to helices 12 with the site of mutation as well as helices nearby, 11 and 13. (D) Magnification of B to highlight the region comprising helices 11–12–13. The mutated residue is depicted as spheres with the carbon in yellow and nitrogen and oxygen colored in blue and red, respectively. (E) Magnification of the region comprising the N370mutation. The blade inserted into SEH1 (light purple) by NUP85 is depicted in blue. The site of mutation is represented as spheres and colored as in (D). (F) Blow up of a slightly rotated NUP43 and its binding region of NUP85. Coloring as in D. The dashed red lines indicate a missing loop and the site of mutation is shown as spheres (colored as in B).
- Bui, K.H., , von Appen, A., DiGuilio, A.L. ,Ori, A., Sparks,L., Mackmull, M-T., Bock, T., Hagen, W., Andrés-Pons, A., Glavy, J.S., Beck, M. (2013). Integrated structural analysis of the human nuclear pore complex scaffold. Cell 2013 Dec 5;155(6):1233-43.doi: 10.1016/j.cell.2013.10.055. [Abstract]
- Dickmanns, A., Kehlenbach, RH. and Fahrenkrog B. (2015). Nuclear Pore Complexes and Nucleocytoplasmic Transport: From Structure to Function to Disease. Int Rev Cell Mol Biol. 320:171-233. [Abstract]
- Port, SA., Monecke, T., Dickmanns, A. Spillner, C., Hofele, R., Urlaub, H., Ficner, R. and Kehlenbach, RH. (2015) ,. Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export. Cell Rep. Oct 27; 13(4): 690 - 702 [Abstract]PDB: [5DIS]
- Monecke, T., Dickmanns, A. Weiss, MS., Port, SA., Kehlenbach, RH. and Ficner R. (2015). Combining dehydration, construct optimization and improved data collection to solve the crystal structure of a CRM1-RanGTP-SPN1-Nup214 quaternary nuclear export complex. Acta Crystallogr F Struct Biol Commun. Dec 1;71(Pt 12):1481-1487. [Abstract] PDB: [5DIS]
- Tauchert, MJ., Hémonnot, C., Neumann, P., Köster, S., Ficner, R. and Dickmanns A. (2016) Impact of the crystallization condition on importin-? conformation. Acta Crystallogr D Struct Biol. 72:705-717. [Abstract] PDB: [4XRI] [4XRK]
- Bonnin, E., Cabochette, P., Filosa, A., Jühlen, R., Komatsuzaki, S., Hezwani, M., Dickmanns, A. Martinelli, V., Vermeersch, M., Supply, L., Martins, N., Pirenne, L., Ravenscroft, G., Lombard, M., Port, S., Spillner, C., Janssens, S., Roets, E., Van Dorpe, J., Lammens, M., Kehlenbach, RH., Ficner, R., Laing, NG., Hoffmann, K., Vanhollebeke, B. and Fahrenkrog, B. (2018) , Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence. PLoS Genet. Dec 13;14(12):e1007845. doi: 10.1371/journal.pgen.1007845. [Epub ahead of print] [Abstract]
- Shaikhqasem, A., Dickmanns, A., Neumann, P. and Ficner R. (2020)Characterization of inhibition reveals distinctive properties for human and Saccharomyces cerevisiae CRM1. J Med Chem.Jul23;63 (14): 7545-7558. [ Abstract] PDB:[6TVO]
- Ravindran, E. , Jühlen, R., Vieira-Vieira, C.H., Ha, T., Salzberg, Y., Fichtman, B., Luise-Becker, L., Martins, N., Picker-Minh, S., Bessa, P., Arts, P., Jackson, M.R., Taranath , A., Kamien , B., Barnett, C., Li, N., Tarabykin, N., Stoltenburg-Didinger, D., Harel, A., Selbach, M., Dickmanns, A., Fahrenkrog, B., Hu, H., Scott, H., and Kaindl, A.M. (2021). Expanding the phenotype of NUP85 mutations beyond nephrotic syndrome to primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders. Hum Mol Genet. 2021 Jun 25; ddab160. doi: 10.1093/hmg/ddab160. Online ahead of print. [Abstract]