Nuclear Pore Membrane Proteins Self-Assemble into Nanopores

Jan 3, 2019 - Large multiprotein nanopores remain difficult to reconstitute in vitro, such as, for instance, the nuclear pore complex (NPC) that regul...
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Nuclear Pore Membrane Proteins Self-Assemble into Nanopores Radhakrishnan Panatala, Suncica Barbato, Toshiya Kozai, Jinghui Luo, Larisa E. Kapinos, and Roderick Y.H. Lim Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01179 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

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Biochemistry

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores Radhakrishnan Panatala1, Suncica Barbato1, Toshiya Kozai1, Jinghui Luo2, Larisa E. Kapinos1 and Roderick Y. H. Lim1,* 1Biozentrum 2Paul

and the Swiss Nanoscience Institute, University of Basel, 4056 Basel, Switzerland

Scherrer Institute, 5232 Villigen, Switzerland

author: [email protected] Phone: +41 61 207 2083; Fax: +41 61 207 2109 *Corresponding

ABSTRACT: Large multi-protein nanopores remain

difficult to reconstitute in vitro, such as for instance the nuclear pore complex (NPC) that regulates macromolecular transport between the nucleus and cytoplasm in cells. Here, we report that two NPC pore membrane proteins self-assemble into ~20 nm-diameter nanopores following in vitro reconstitution into lipid bilayers. Pore formation follows from the assembly of Pom121 and Ndc1 oligomers, which arrange into ring-like membrane structures that encircle aqueous, electrically conductive pores. This represents a key step towards reconstituting membrane-embedded NPC mimics for biological studies and biotechnological applications.

Membrane-embedded protein nanopores are important in several technologies such as DNA sequencing1,2 and protein biosensing3,4. These typically employ pore-forming toxins that comprise of β-barrel proteins (e.g., α-hemolysin) or α-helical barrels (e.g., ClyA) that present narrow channel diameters of 90% of all GPLs were stained with Nile Red, a lipid membrane stain. Among those, we followed the GFP signal on Ndc1 upon its standalone expression or co-expression with Pom121 (Figure 2c). This yielded Ndc1-eGFP signal intensities of ~30% for Pom121-Ndc1-GPLs and ~10% for Ndc1-GPLs. The control blank giantliposomes (GLs) gave null results (Figure 2c). At the nanoscale, high-speed atomic force microscopy (HS-AFM) revealed numerous pore-like perforations in the Pom121-Ndc1-PL membrane (Figure 3a) that were not present in blank liposome controls (Figure S2). Interestingly, each pore is enclosed by a ring-like structure above the membrane surface (Figure 3b). Each ring suggests that individual Pom121-Ndc1 sub-complexes assemble into a corral-like arrangement. Their average pore dimensions are 5.8 ± 1.4 nm, 19.5 ± 3.7 nm and 37.3 ± 6.1 nm for height (Hpore), inner diameter (i.d.) and outer diameter (o.d.), respectively (Figure 3b). Moreover, the ring height (Hrim) and thickness of the surrounding membrane (Hmem) as measured from the top of each ring and bottom of each pore to the membrane surface are 3.2 ± 1.4 nm and 2.5 ± 0.6 nm, respectively. Here, Hmem appears smaller than the expected lipid bilayer thickness37. This may be due to a limited penetration of the HS-AFM tip into the pore or that Pom121-Ndc1 pore formation might cause a local deformation (compression) of the lipid bilayer

surrounding it38. In addition, we sometimes observed features that resembled elongated pores (or “slits”) (Figure 3c) that were 107.7 ± 21.6 nm in length (Figure S3) but with similar Hpore, i.d. and o.d. values (Figure 3D). This suggests that the slits might form from conjoined pores. Figure 4: Ascending staircase-like conductance behavior indicates that the numbers of Pom121-Ndc1 pores are increasing over time. Each upward “step” signifies the emergence of a new pore but local fluctuations indicate stochastic behavior during protein insertion. As controls, blank liposomes, Pom121-PLs and Ndc1-PLs did not form pores.

Finally, we applied single channel electrical measurements39 to verify that the TM Nups formed aqueous pores that punctured the lipid membrane (Figure 4). In the absence of pores, such as in blank liposomes, the membrane exhibits a property of zero conductance. Similar results were obtained for Pom121- or Ndc1-only PLs, which did not show any evidence of pore formation. In marked contrast, Pom121-Ndc1-PLs exhibited an overall staircaselike behavior40 that ascended until the detection limit of the instrument was reached. Importantly, each upward “step” signifies the emergence of a new pore although local fluctuations suggest more complex behavior41 such as the formation, closure, and fusion of multiple pores. Moreover, each step can be used to estimate a theoretical pore size42 giving 3.8 ± 2.0 nm, which is in approximate agreement with our HS-AFM results. For completeness, pore formation did not occur when blank liposomes were added to Pom121-PLs and Ndc1-PLs respectively, nor during sequential additions of Ndc1-PLs to Pom121-PLs (Figure S4). However, the electrical signal destabilized when Pom121-PLs were introduced to Ndc1-PLs, which may suggest that Pom121 facilitates a rearrangement of Ndc1 in the membrane. Still, how the sequential addition of Pom121 and Ndc1 favors pore formation remains to be ascertained.

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Biochemistry

AUTHOR INFORMATION Corresponding Author *[email protected]

Funding Sources

No competing financial interests have been declared.

To summarize, our work presents Pom121-Ndc1 pores in lipid membranes. Their diameters are smaller than those of NPCs, as NPCs are formed by fusing the inner and outer nuclear membranes26 whereas Pom121-Ndc1 pores are embedded in lipid bilayers (Figure S5). More in-depth studies will be required to resolve the structure and orientation of each protein, as well as how they self-assemble and from which side of the PL membrane the ring structures emanate i.e., luminal or external. Furthermore, the Pom121 FG domain binds Kapβ1 and the RanGDP importer nuclear transport factor 2 (NTF2) with a binding affinity of 71 ± 15.4 nM and 2.77 ± 0.31 µM, respectively (Figure S6). Hence, Pom121-Ndc1 pores may be able to facilitate the selective transport of NTRs to serve as NPC mimics. We further envisage that the Pom121-Ndc1 pores may be used as templates to build on other NPC components that interact with Pom121 and Ndc128. This opens up new strategies to de novo engineer NPC-based nanopores for biological studies and diverse biotechnological applications43.

R.P. and S.B. are supported by the National Centre of Competence in Research in Molecular Systems Engineering. T.K. is supported by a Swiss Nanoscience Institute Fellowship. Further support to R.Y.H.L. is provided by a Swiss National Science Foundation grant no. 31003A_170041.

ACKNOWLEDGMENT We thank Thomas K.C. Bock and Alexander Schmidt from the Proteomics Core Facility, Janine Bögli from the FACS Facility and Yusuke Sakiyama for advice and technical assistance with HS-AFM.

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ASSOCIATED CONTENT Supporting Information. Supplementary Figures for Figures 1, 3 and 4, and Supplementary Figures S5 and S6 with their respective figure legends. Excel File showing mass spectrometry data and analysis. Materials and Methods.

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