The Nup62 Coiled-Coil Motif Provides Plasticity for Triple-Helix Bundle

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Article

Nup62 coiled-coil motif provides plasticity for triple helix bundle formation. Pravin S. Dewangan, Parshuram J. Sonawane, Ankita Rai Chouksey, and Radha Chauhan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01050 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 15, 2017

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Biochemistry

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Nup62 coiled-coil motif provides plasticity for triple helix bundle formation. Pravin S. Dewangan#, Parshuram J. Sonawane#, Ankita R. Chouksey, Radha Chauhan* National Centre for Cell Science, S. P. Pune University Campus, Ganeshkhind, Pune 411007, India.

#

indicate equal contributions from the authors *Corresponding Author address: Radha Chauhan, National Centre for Cell Science, S.P. Pune University Campus, Ganeshkhind, Pune 411007, Maharashtra, India. Email: [email protected] Phone: +91-20-25708255 Fax:+91-20-2569 2259

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ABBREVIATIONS NPC, Nuclear Pore Complex; Nup, Nucleoporin; SEC, Size Exclusion Chromatography; MALS, Multi-Angle Light Scattering; IPTG, Isopropoyl β-D-1-thiogalactopyranoside; DTT, Dithiothreitol ; EDTA, ethylene diamine tetra acetic acid; PEG, poly-ethylene glycol; XRD, X-ray diffraction; Ni-NTA, nickel-Nitrilo triacetic acid; SDS-PAGE, sodium dodecyl sulphate poly-acrylamide gel electrophoresis ; RI, refractive index; ExPASy, expert protein analysis system ; RMSD, root mean square deviation. CCP4, Collaborative Computational Project Number 4; PyMOL, Python-enhanced Molecular Visualization Tool, COOT, Crystallographic Object-Oriented Toolkit; XDS, X-ray Detector Software, PHENIX, Python-based Hierarchical Environment for Integrated Xtallography.


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ABSTRACT

The central transport channel of vertebrate nuclear pore complex (NPCs) is made up of nucleoporins: Nup62, Nup54 and Nup58. The coiled-coil domains in α-helical regions of these nucleoporins are thought to be crucial for several protein-protein interactions in the NPC sub-complexes. In this study, we determined the crystal structure of the coiled-coil domain of rat Nup62 fragment (362-425) to 2.4 Å resolution. The crystal structure shows conserved coiled-coil domain as a parallel three helix bundle for the Nup62(362-425) fragment. Based on our size exclusion chromatography coupled to multi-angle light scattering analysis and glutaraldehyde crosslinking experiments, we conclude that the Nup62(362-425) fragment display dynamic behavior in solution and can also exist either in homodimeric or homotrimeric states. Our comparative analysis of the rat Nup62(362-425) homotrimeric structure with previously reported heterotrimeric structures [rat Nup62(362-425)-Nup54(346407)

and

Xenopus

Nup62(358-485)-Nup54(315-450)-Nup58(283-406)

complexes]

demonstrate the structural basis for parallel triple helix bundle formation for Nup62 with different partners. Moreover, we show that the coiled-coil domain of the Nup62 is sufficient to interact with the coiled-coil domain of rat Exo70, a protein in exocyst complex. Based on these observations, we suggest the plausible chain replacement mechanism that yields to diverse protein assemblies with Nup62. In summary, the coiled-coil motif present in Nup62 imparts the ability to form a homotrimer and heterotrimers either with Nup54 or Nup54Nup58 within the NPCs as well as with Exo70 beyond the NPCs. These complexes of Nup62 suggest the crucial role of the coiled-coil motifs in providing the plasticity to various modular assemblies.


Biochemistry

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Bidirectional transport of the cargo between nucleoplasm and cytoplasm occurs through the nuclear pore complexes (NPCs), multi-protein assemblies that are embedded in the nuclear envelope. These NPCs are made up of approximately 30 distinct proteins called nucleoporins (Nups) that are classified into various sub-complexes. The central transport channel (CTC) is one of such complexes located at the central region of the NPC and is important site for the shuttling of specific cargoes across the NPC. The CTC of the mammalian NPC is comprised of four nucleoporins: Nup62, Nup54, Nup58 and Nup45 (a splice variant of Nup58)1. These Nups contain repeats of Phenylalanine and Glycine, referred as FG repeats, that act as docking sites for incoming and outgoing karyopherin bound cargoes2. Earlier studies in rat have shown that these CTC Nups contain relatively smaller structured regions, mostly comprising the α-helical domains that are essential for the formation and function of CTC complex2, 3. The structured regions of CTC Nups interact with Nup93 of the adapter sub-complex and is crucial for holding CTC at the central plane of the NPC for proper transport functions4-6. The carboxy-terminal α-helical regions of CTC Nups harboring canonical coiledcoil motifs are shown to be important for protein-protein interactions5,

7, 8

. Generally, the

coiled-coil domains are structural motifs in which two to five α-helices are wrapped around each other to form a helical bundle or a supercoil. Most commonly occurring coiled-coil domains are usually left handed supercoils having periodicity of 7 with ~3.6 residues per turn9-12. This gives the α-helices a unique property of having the ‘i + 7th’ amino acid on the same bundle axis as ‘ith’ and ‘i + 14th’ amino acids, that forms a heptad repeat. These heptad repeats can be represented as the repeat patterns of ‘a-b-c-d-e-f-g’ on each of the α-helices13, 14

. Positions ‘a’ and ‘d’ are mostly non-polar residues, while positions ‘e’ and ‘g’ are polar

ones that may form the salt bridges; positions ‘b’, ‘c’ and ‘f’ are mostly solvent exposed


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hydrophilic amino acids

15

. Largely, the coiled-coil motifs are predicted to be important for

protein-protein interactions in various NPC sub-complexes and several other proteins 7, 15-23. These well conserved coiled-coil motifs (across eukaryotes) in Nup62, Nup54 and Nup58

are

crucial

for

holding

the

central

channel

assembly

and

form

the

Nup62•Nup54•Nup58 complex (also referred as CTC complex)24. Recent structure of Xenopus CTC complex shows that partial helical regions of the Nup62(358-485)•Nup54(315450)•Nup58(283-406) complex are wrapped around each other forming two helical bundles25. The CTC Nups in yeast Nsp1, Nup57 and Nup49 are homologs of Nup62, Nup58 and Nup54, respectively and a structure of CTC complex from thermophilic yeast (Chaetomium thermophilum),

Nsp1(463-678)•Nup57(77-326)•Nup49(246-470)•Nic96(139-180)

have

showed three helical bundles with coiled-coil domains and one of the domains interacting with two short helices of the Nic96(139-180) region26. Moreover rat Nup62(362425)•Nup54(346-407) complex forms coiled-coil heterotrimer with one α-helix of Nup54(346-407) wrapped around two α-helices of Nup62(362-425)18. Nup62 is also a part of another complex with Nup214 (Nup159 in yeast) and Nup88 (Nup82 in yeast) in the NPC7, 16. Electron microscopic analysis of this complex from the yeast shows three distinct coiled-coil domains in the complex of Nup159•Nup82• Nsp116. These structures suggest a crucial role of coiled-coil motifs in the formation of various complexes of Nup62. Interactions between mammalian CTC Nups are dynamic in solution17-19, 27, 28, as it is demonstrated by the formation of in vitro complexes of homotetrameric Nup54(453-494)17 and Nup58(327-415)19, heterotrimeric Nup54(346-407)•Nup62(362-425)18 or heterooligomeric

Nup58(327-411)•Nup54(456-494)18.

Since,

the

coiled-coil domains

are

unambiguously present in these Nups, we postulated their role in providing plasticity to the CTC complexes.


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Additionally, mammalian Nup62 is involved in several cellular processes outside the NPCs29-31 indicating the possibility of its interactions with various other cytoplasmic proteins. For example, α-helical region of the Nup62 interacts directly with Exo70 to regulate cell migration and actin dynamics in conjugation with Exo70, which is a part of the exocyst complex29. Structural role of the coiled-coil domains of Nup62 in such interactions remains poorly understood. In this study, we report the crystal structure of rat Nup62(362-425) protein at 2.4Å resolution. We found that Nup62(362-425) is a homotrimer and contains structurally conserved coiled-coil motif (371-391), which is important for different assemblies of Nup62 complexed with various Nups. Our crosslinking and SEC-MALS experiments show that Nup62 shows dynamic behavior in solution with the equilibrium of homodimer and homotrimer. Moreover, we observed that the coiled-coil motif of rat Nup62(362-425) in αhelical region is important for interactions with rat Exo70, suggesting the importance of such motifs for interactions with proteins other than Nups. Overall this comparative structural analysis of rat Nup62(362-425) protein enhances our understanding of the role of conserved coiled-coil motif in providing plasticity to form various assemblies within NPC and beyond.


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MATERIALS AND METHODS Purification of rat Nup62(362-425) protein fragment Rat Nup62(362-425) was cloned in pET28a vector and expressed in E. coli BL21(DE3)-RIL cells, by induction with 0.2 mM IPTG (MP Biomedicals) at 30°C for 3 to 4 hours. His-6x-tagged rat Nup62(362-425) was purified using Ni-NTA agarose beads (Qiagen). The eluted protein was digested with thrombin (Calbiochem) during dialysis at 4°C. The protein was concentrated using Amicon Ultra with 3 kDa cut off (Merck) and subjected to SEC using HiLoad 16/600 Superdex 200pg 16/60 column (GE Healthcare) in SEC buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA and 1 mM DTT; adapted from ref. 19). Four different concentrations of purified rat Nup62(362-425) were used for the oligomerization analysis using Superdex 200 10/30 GL column (GE Healthcare) in SEC buffer. Protein crystallization 18 mg/ml protein concentration was used for vapor diffusion hanging drop protein crystallization. Hampton Crystal screen I & II (Hampton Research) was used for screening. The Initial condition (100 mM Tris-HCl pH 8.0 to 8.5, 100 mM MgCl2, 30-38 % V/V PEG 400) was fine tuned to obtain diffraction quality crystals. The final condition was: 100 mM Tris-HCl pH 8.0-8.5, 50 mM MgCl2, 34 % V/V PEG 400. 0.5-2% Glycerol was used as an additive to get single crystals. All crystallization experiments were performed at 18°C. Data Collection and Structure Determination The cryo-condition optimization was done by soaking crystals in buffer containing 100 mM Tris-HCl pH 8.0 to 8.5, 50 mM MgCl2, 34% V/V PEG 400 and 8% glycerol for 2 to 5 minutes. X-ray diffraction data from single crystal was collected at XRD1 beamline, Elettra Sincrotrone, Trieste (Italy) at 0.98 Å and processed using X-ray Diffraction Software (XDS)32. The data was scaled using ‘scala’ from CCP4 suite33. Molecular replacement was


Biochemistry

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performed using one of the Nup62 chains from rat Nup62(362-425)•Nup54(346-407) complex (Ref. 18, PDB ID: 3T97) as a template in PHENIX. Initial model was obtained through Autobuild in PHENIX34. It was further manually built using COOT35 and refined using Refine in PHENIX34. Analysis of rat Nup62 (362-425) Structure The structure and interface analysis was done using PyMOL36 and PISA37. The solvation energy for the molecule was calculated in PISA. LSQ Superimposition was performed in COOT using the C-alpha atoms. All the images were prepared using PyMOL. The helical wheel representation was prepared using Drawcoil 1.038. The propensity of full length proteins to form coiled-coil motifs were calculated using COILS server from ExPASy12 and MARCOIL39. In COILS program, three windows of residues 14, 21 and 28 were used independently to calculate the propensity. The interface of rat Nup62(362-425) homotrimer was visualized in PyMOL using output script from the SOCKET40. Cross-linking of rat Nup62(362-425) The protein was purified as mentioned previously except 10 mM Tris pH 8.0 was replaced with 50 mM phosphate buffer, pH 8.0. The protein crosslinking was performed according to Fadouloglou et al, ref. 41. Briefly, 200 µL of the protein at two different concentrations (0.5 mg/mL and 5.85 mg/mL) were allowed to bind to 0.5 mL Ni-NTA agarose beads (Qiagen) and washed with wash buffer (50 mM phosphate buffer pH 8.0, 150 mM NaCl, 10 mM Imidazole pH 8.0). Crosslinking solution (50 mM phosphate buffer pH 8.0, 150 mM NaCl, 0.1% glutaraldehyde) was passed over the protein bound beads. The protein samples were eluted in the elution buffer (50 mM phosphate buffer pH 8.0, 150 mM NaCl, 300 mM Imidazole pH 8.0,). Equal amounts of cross-linked proteins were then loaded onto 10-20% gradient Tricine Protein Gel (Novex; Life Technologies) and analyzed by reducing SDS-PAGE followed by coomassie staining.


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Size exclusion chromatography coupled Multi-angle light scattering (SEC-MALS) analysis SEC-MALS for the purified rat Nup62(362-425) protein was performed on Superdex 200 10/300GL (GE Healthcare) connected to Agilent HPLC system equipped with the 18-angle light scattering detector (Wyatt Dawn HELIOS II) and the refractive index detector (Wyatt Optilab T-rEX). The system was calibrated with the BSA at a concentration of 2 mg/mL. 100µL of two different concentrations (18 mg/mL and 5.85 mg/mL) of the proteins were injected and molecular weight was calculated using ASTRA software (Wyatt Technologies). Modelling and Validation of rat Nup62(362-425)-Exo70(1-100) interactions Since, it is reported previously that the N-terminal region (1-75) of Exo70 might have coiled-coil regions 42, PrOCoil43 prediction was performed for the first 100 residues of rat Exo70. In order to generate a homology model, one of the chains of rat Nup62(362-425) homotrimer (as described above, PDB: 5H1X) was mutated to a residue range 33-81 of rat Exo70 in COOT and was corrected using ‘geometry minimize’ from PHENIX. LSQ superimposition of the two structures was performed in the COOT. The images were prepared using PyMOL. To validate the interaction of Nup62 with Exo70, the α-helical region of rat Nup62 (323-525) was cloned into pET28a vector. Rat Exo70 full length (1-653) and its deletion fragments (1100, 100-384) were cloned in pGEX-4T1 (GE Healthcare) and all the constructs were confirmed by DNA sequencing. These deletion constructs were designed based on Bao et al, ref 44. The sequences of the GST-fusion protein variants used for this study are mentioned in the supplementary Figure S7. Both Nup62 and Exo70 fragments harboring plasmids were cotransformed into E. coli BL21(DE3)-RIL cells. The cultures were induced by 0.2 mM IPTG at 18 °C for 12 hours, followed by Ni-NTA affinity based pull downs of the soluble cell


Biochemistry

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lysate. The eluted fractions were separated by SDS-PAGE in the tricine buffer and western blotting was performed using anti-GST-HRP conjugate (Invitrogen Technologies). Signals were recorded using Imager600 (GE Healthcare).


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RESULTS Coiled-Coil propensity of rat Nup62, Nup58 and Nup54 proteins All the three CTC Nups have two major domains, flexible unstructured FG repeat region and structured α-helical region (Figure S1). We see that the structured region shows the coiled-coil propensity for all the three Nups (Figure S1). At the window size of 28 in COILS, Nup62 helical region has three distinct peaks and residues between 366 and 423 show highest coiled-coil propensity. Rat Nup54 has highest coiled-coil propensity for 346407 region followed by 453-500 region. Nup58 has highest propensity for coiled-coil formation for 327-415 region. (Figure S1). We also observed similar predictions for all three Nups when MARCOIL39 program was used. Crystal Structure of rat Nup62(362-425) The structure of rat Nup62(362-425) was solved in space group P21 with the unit cell parameters of a = 30.98 Å, b = 97.56 Å, c = 31.50 Å, α = γ = 90°, β = 112.85°. The data was processed upto 2.4 Å using XDS. The molecular replacement solution showed that there are three molecules of Nup62(362-425) in the asymmetric unit. The residues that were built by autobuild in PHENIX were corrected in COOT and remaining residues were added manually using COOT graphics software. The structure was refined to a resolution of 2.4 Å with Rwork of 22.69 and Rfree of 30.77. The model was validated by Ramachandran analysis45 in COOT and PHENIX. After several rounds of the refinement in PHENIX, the electron density was not observed for the last 17 residues of C- terminus of chain A and C and 13 residues for the chain B. Table 1. Data Processing and Refinement Statistics:


Biochemistry

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Space Group = (4) P 1 21 1 Average Mosaicity = 0.21 Maximum Resolution: 2.4 Å Average Unit Cell

a

b

c

α

β

γ

30.98

97.56

31.50

90.00

112.85

90.00

Low resolution limit High resolution limit Rmerge Total number of observations Total number unique Mean (I / sd (I)) CC(1/2) Completeness Multiplicity

Overall 48.78 2.41

Inner Shell 48.78 7.62

Outer Shell 2.54 2.41

0.039

0.026

0.176

22313

744

3034

6669 17.9 0.999 99.4 3.3

222 38.5 0.999 98.9 3.4

934 5.9 0.982 98.6 3.2

Refinement Statistics Resolution (Å) No. of reflections Rwork/ Rfree Number of non-hydrogen atoms Protein Water Average B-factors Protein Water

27.82- 2.41 6650 0.2269/0.3077 1239 1228 11 54.6 54.7 53.4

R.M.S deviations Bond lengths (Å) Bond angles (º)

0.008 1.120

Ramachandran Statistics favoured Outliers allowed C-beta Outliers Clash Score

96.4% 0.7% 1.43% 0 7.33

The asymmetric unit contains three chains of rat Nup62(362-425) fragment intertwined with each other forming a coiled-coil packing (Figure 1a). These three helices run parallel to each other and contain majorly hydrophobic amino acids at the interface. The overall structure of this fragment is very similar to rat Nup62(362-425)•Nup54(346-407) complex as reported previously18 (Figure S2). The interface of the coiled-coil packing has continuous seven layers of hydrophobic residues interacting with each other from all three chains, with exception of one glutamine


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(Q389) (Figure 1b & 1c). The helical wheel plot for Nup62 sequence show non-polar residues at ‘a’ (I375, V382, L396) and ‘d’ positions (L378, V385, L392) forming the interface. Nup62 amino acids E381 and R391 from one chain form two salt bridges with K386 and D393 of another chain, respectively (Figure 1c).

Figure 1. The crystal structure of rat Nup62(362-425)

N-terminal

75 Å

C-terminal

(a)

(b)

(c)

Figure 1: The crystal structure of rat Nup62(362-425). (a) The structure of rat Nup62(362425) is a coiled-coil parallel homotrimer. (b) The interface of rat Nup62 homotrimer prepared using SOCKET. ‘Knob-into-holes packing’ region is shown in green color with residues at interface in cyan color are shown as sticks. Rest of helices are shown as grey ribbons (c) Helical wheel projection for Nup62 sequence (residues 375-396) in the coiled-coil domain. Amino acid residues depicted in red, blue, grey and orange circles are negatively charged, positively charged, non-polar, polar residues, respectively. Red dash lines indicate ionic interactions between the helices.

In Solution Oligomeric Status of rat Nup62(362-425) Size exclusion chromatography of the purified rat Nup62(362-425) protein using Superdex200(10/30)GL column at various concentrations (24mg/mL, 18 mg/mL and 1.98


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mg/mL and 0.43mg/mL) showed a lowering of elution volume peak with increasing concentration of the protein (Figure 2a). At lower concentration (0.43 mg/mL) the protein elutes at the peak volume of 15.9 mL, and at 1.98 mg/mL elutes at 15.3 mL (Figure 2a; highlighted by blue and green arrows in the chromatogram). While at higher concentrations (18 and 24 mg/mL) it elutes at 14.9 mL as a major peak along with a shoulder (Figure 2a; highlighted as orange/red arrow). These observations clearly indicated concentration dependent oligomerization changes in the Nup62(362-425) protein. To further validate, we performed size exclusion chromatography coupled to multiangle light scattering (SEC-MALS) at two concentrations (5.85 and 18 mg/mL) that showed shift in the molecular weights of peak from 11.7 kDa (for 5.85 mg/mL) to 22.43 kDa (for 18mg/mL) corresponding to trimer (Expected molecular mass of monomer is 7.5 kDa; Figure S3). Thus, suggesting the dynamic equilibrium of the mass range in the purified Nup62(362425) protein. To confirm this oligomerization dynamics, we performed experiments with two concentrations of the protein (0.5 and 5.85 mg/mL) for on-column (Ni-NTA beads) glutaraldehyde crosslinking41. The lower concentrations of the protein are expected to have less molecular crowding environment, therefore likely to reduce crosslinking of non-specific oligomers. The SDS-PAGE analysis of the eluted crosslinked proteins showed appearance of approximately 20 kDa and 30 kDa bands along with ~10 kDa band (Figure 2b), corresponding to dimer, trimer and monomer, respectively (expected molecular weight of the monomer is 9.8 kDa with His-6x-tag). Similar patterns of crosslinking were observed with both concentrations of the protein. However, the amount of homodimers and homotrimers were seen higher at 5.85 mg/mL (Figure 2b). This suggests that the Nup62(362-425) protein at higher concentration has higher population of the homodimers and the homotrimers compared to the lower concentration.


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Figure 2. Oligomerization studies of rat Nup62(362-425):

Figure 2: Oligomerization studies of rat Nup62(362-425). (a) Gel filtration analysis of rat Nup62(362-425) on Superdex 200 10/300GL column at four different concentrations of 24 mg/mL (red), 18 mg/mL (orange), 1.98 mg/mL (green) and 0.43 mg/mL (blue). The shift in peak maxima is highlighted by arrows (b) Tricine SDS-PAGE scan showing rat Nup62(362425) crosslinked at concentrations of 0.5 mg/mL and 5.85 mg/mL on Ni-NTA resin using 0.1% glutaraldehyde solution. The cross-linked band can be seen at 20 kDa dimer and 30 kDa trimer in both concentrations. The 30 kDa band is more prominent at 5.85 mg/mL.

Our crosslinking analysis is in agreement with the observation of concentration dependent shift in the molecular weights in Nup62(362-425) protein as determined by SEC


Biochemistry

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and SEC-MALS analysis. Our SEC-MALS data does not rule out the possibility of monomeric-dimeric equilibrium (Mw 11.7 kDa) particularly at the lower protein concentration (Figure S3). However, the equilibrium seems to shift towards dimeric-trimeric state at the higher protein concentration (Figure S3). Therefore, we conclude that the Nup62(362-425) protein predominantly shows homodimer-homotrimer dynamics.

Comparison of rat Nup62(362-425) homotrimer, rat Nup62(362-425)•Nup54(346-407) heterotrimer

Xenopus

and

Nup62(358-485)•Nup54(315-450)•Nup58(283-406)

heterotrimer The overall architecture of rat Nup62(362-425) homotrimer is similar to the corresponding regions in the heterotrimer of rat Nup62(362-425)•Nup54(346-407)(PDB ID: 3T97; Figure S3) and Xenopus Nup62(358-485)•Nup54(315-450)•Nup58(283-406) complex (PDB ID: 5C3L; Figure S4). Both rat and Xenopus heterotrimeric structures have parallel three helix bundle with a coiled-coil packing in the region corresponding to rat Nup62(362425). The superimposition of rat Nup62(362-425) homotrimer with rat Nup62(362425)•Nup54(346-407) heterotrimer showed RMSD of 1.93 Å (Figure S3). Similarly, the superimposition

of

rat

Nup62(362-425)

homotrimer

with

Xenopus

Nup62(358-

485)•Nup54(315-450)•Nup58(283-406) complex showed 1.94 Å RMSD (Figure S3). This analysis clearly indicates that despite of differences in the sequences of Nup62, Nup58 and Nup54, the overall architecture of the parallel three helix bundle is preserved.

Comparison of rat Nup62(362-425) homotrimer vs rat Nup62(362-425)•Nup54(346-407) heterotrimer: It appears that the topology of Nup62(362-425) homotrimer and rat Nup62(362425)•Nup54(346-407) heterotrimer are similar yet there are significant differences in the ionic interactions between the chains. There are more number of salt bridges (eight in the


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heterotrimer vs six in the homotrimer) in the Nup62(362-425)•Nup54(346-407) heterotrimer as compared to that of Nup62(362-425) homotrimer (Table S1). The two salt bridges (K386E381 and R391-D393) between the two Nup62 chains within heterotrimer (Figure 3a and 3c) are retained in rat Nup62(362-425) homotrimer (Figure 3b and 3d). Only one pair of residues is available for these salt bridges in Nup62(362-425)•Nup54(346-407) heterotrimer, whereas these salt bridges form a ring of interactions to stabilize Nup62(362-425) homotrimer (Figure 3b and 3d). Apart from the above mentioned salt bridges, Nup62(362-425)•Nup54(346-407) heterotrimer also has E407 and E414 from Nup62(362-425) that interacts with K381 and R388 from Nup54 fragment, respectively18. Interestingly, the amino acids that are involved in the salt bridge formation between Nup62 and Nup54 are not involved in the salt bridge formation in the homotrimer, suggesting a unique set of interactions in the Nup62(362425)•Nup54(346-407) heterotrimer (Table S1).

Figure 3. Comparison of rat Nup62(362-425) homotrimer with rat Nup62(362425)•Nup54(346-407) heterotrimer:


Biochemistry

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Figure 3: Comparison of rat Nup62(362-425) homotrimer with rat Nup62(362425)•Nup54(346-407) heterotrimer. Amino acids involved in salt bridges are shown as sticks, with color code: red – oxygen, blue - nitrogen, grey/green –carbon and yellow dotted line represent salt bridges. Numbers above dotted line indicate distance between involved salt bridges residues. Ionic interactions of (a) Glu381(chainA)-Lys386(chainC) from rat Nup62(362-425)•Nup54(346-407) heterotrimer is compared with (b) Glu381-Lys386 from rat Nup62(362-425) homotrimer and (c) Arg391(chainA)-Asp393(chainC) in rat Nup62(362425)•Nup54(346-407) heterotrimer compared with (d) Arg391-Asp393 of rat Nup62(362425) homotrimer.

Despite of low sequence similarity between rat Nup54 and Nup62, the hydrophobic core

in

both

Nup62(362-425)

homotrimer

and

Nup62(362-425)•Nup54(346-407)

heterotrimer are comparable. This has been accomplished due to positioning of the hydrophobic residues at the ‘a’ and ‘d’ position (Figure 1b-c & S5a) of the coiled-coil motif


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of both structures18. Similar arrangement of hydrophobic resides is also observed in the

Xenopus CTC sub-complex (Figure S5b). The PISA analysis also showed that there are significant differences in the stability of homotrimer and heterotrimer (Table S2). All three interfaces of Nup62(362-425)•Nup54(346-407) complex showed a complex formation significance score (CSS) of 1.0 implying that interactions between the three chains are strong. While the CSS for interfaces in the homotrimer (between Chain B – Chain A, Chain A – Chain C and Chain B – Chain A) are 0.659, 0.341 and 0.659, respectively (Table S2). This implies that the affinity of the three chains in homotrimer is lower as compared to Nup62(362-425)•Nup54(346-407) chains. The interface score between Chain A and Chain C in homotrimer is the lowest (0.341) suggesting that one of these chains can easily dissociate from the homotrimer. This observation is in coherence with our interpretation of SEC, SECMALS and glutaraldehyde crosslinking experiments that Nup62(362-425) homotrimer can exist in dynamic equilibrium with the homodimer. It is interesting to observe that all ionic interactions between Nup62 and Nup54 in rat Nup62(362-425)•Nup54(346-407) are conserved in the corresponding Xenopus structure. On the other hand, Nup58 has very few ionic interactions either with Nup54 or Nup62 in the long helix bundle (Table S1). Arrangement of Glutamine in the interface of coiled-coil triple helix bundle The interface of both rat Nup62(362-425) homotrimer and Nup62(362425)•Nup54(346-407) heterotrimer is made up of mostly hydrophobic residues with the exception of glutamine at position 389 in Nup62. In Nup62(362-425)•Nup54(346-407) heterotrimer, amino group from Q386 of Nup54 chain forms a polar interaction with carbonyl group of Q389 of chain C and with a main chain carbonyl group of 393rd residue. The amino group of Q389 of chain A (Nup62) forms salt bridge with carbonyl group of Q389 of chain C (Nup62). The same Q389 interacts with the main chain carbonyl group of 390th residue of


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Nup54 fragment (Figure 4a). In Nup62(362-425) homotrimer the Q389 are in similar orientations but beyond the distances for hydrogen bonding. There is no interaction between the glutamines in Nup62(362-425) homotrimer (Figure 4b). In comparison of the glutaminelayer (Q-layer) of rat Nup62(362-425) homotrimer with Xenopus CTC sub-complex, we see that Q-layer interaction is lost as Nup58 has Leucine at the 389th position (Figure 4c). Thus, it is prominent that the Q-layer interactions are stronger only with Nup62 and Nup54 heterotrimer. Figure 4. Comparison of the glutamine-layer in rat Nup62(362-425) homotrimer, Nup62(362-425)•Nup54(346-407) heterotrimer and Xenopus CTC sub-complex:


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Figure 4: Comparison of the glutamine-layer in rat Nup62(362-425) homotrimer, Nup62(362-425)•Nup54(346-407) heterotrimer and Xenopus CTC sub-complex. Residues are represented as sticks. Depicted as red – oxygen; blue - nitrogen; grey, cyan, green, pale green, pale blue and pale orange –carbon atoms and yellow dotted line salt bridge. Numbers above dotted line indicate bond length. (a) Glutamine 389 present in the interface of the three chains of rat Nup62 homotrimer do not interact with each other. (b) The three glutamines from Nup62 (389th position) and Nup54 (368th position) interact with each other forming a network of ionic interaction. (c) In Xenopus, Nup58 chain has a Leu389, which is a non-polar residue at the interface as compared to glutamine from either Nup54 or Nup62. There are no ionic interactions at the glutamine-layer in Xenopus CTC sub-complex.


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Interactions studies of Nup62 with Exo70: It is shown earlier that Exo70 can interact with Nup62 helical regions29. The ProCoil prediction showed the coiled-coil motif in rat Exo70 is present between 33 and 81 residues (Figure S6a-b). Therefore, we hypothesized that Nup62 and Exo70 interactions might be mediated through the coiled-coil regions. A homology model was generated for corresponding rat Exo70 region and modeled further to generate Nup62(362-425)•Exo70(3381) heterotrimer (Figure S6c). The resulting model upon superimposition showed similar structure with rat Nup62(362-425) homotrimer with RMSD of 1.35 Å (Figure S6d) indicating similarity in the triple helix bundles. To validate these interactions, His-6x affinity based pull down assay was performed between rat Nup62 α-helical regions (323-525 and 362-425) and rat Exo70 α-helical regions (1-100 and 100-384). Since full-length GST-Exo70 (1-653) construct was not soluble in E. coli expression system, this construct was omitted from the study. The Ni-NTA affinity-pulled complexes were analyzed by SDS-PAGE followed by western blot analysis and detected for the Exo70 proteins using anti-GST antibody. This analysis showed that the longer region of Nup62(323-525) is able to pull out GST-Exo70(1100) (Figure 5a). This interaction is consistent with the previous observation of interaction of human Nup62(328-522) with human Exo70(1-393)29. Interestingly, the shorter region of Nup62 was also able to pull out GST-Exo70(1-100) (Figure 5b) suggesting that the coiledcoil regions of both Nup62 and Exo70 are sufficient enough to interact with each other to form a Nup62•Exo70 complex. Figure 5. Validation of Nup62(362-425)•Exo70(1-100) interactions:


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Figure 5: Interaction study of Nup62 with Exo70. Combination of two deletion proteins (323-525 and 362-425) of His-6x-tagged Nup62 and two variants (1-100 and 100-384) of GST-tagged Exo70 were co-expressed and pulled down using Ni-NTA beads and visualized on coomassie stained SDS-PAGE (top panel) as well as western blot using anti-GST antibody (lower panel). E – Elution, I – Input, and red arrow indicates GST-Exo70(1-100), orange arrow-only GST protein. His-6x tagged Nup62(323-525) is indicated by blue arrow and His6x tagged Nup62(362-425) protein band is indicated by green colored arrow.


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DISCUSSION The coiled-coil motifs are known to act as regions of interaction with other proteins. It has been reported that the coiled-coil region of Nup62 interacts with proteins within NPC and beyond16,

29, 46

. Structural comparison of the interactions of Nup62 homotrimer with

Nup62 heterotrimers (formed with Nup54 and Nup58) revealed the basis for three helix bundle formation. Based on our analysis, it seems that Nup62(362-425) is sufficient enough for triple helix assembly in the presence or absence of various interacting partners. Various stoichiometric models for the rat central channel have been proposed3, 24, 4649

indicating the dynamic behavior of the CTC sub-complex. However, biochemical basis of

these stoichiometries is not clear. Our study indicates that the Nup62(362-425) coiled-coil region can easily form homodimer and homotrimer in solution and upon increasing the concentration, equilibrium is shifted towards the homotrimer. The solvation energy of this homotrimer is -38.3 kcal/mol, which is lower than solvation energy of Nup62(362425)•Nup54(346-407) heterotrimer (-50.4 kcal/mol) (Table S2). This implies that the chains of Nup62(362-425) homotrimer would dissociate easily as compared to the Nup62(362425)•Nup54(346-407) heterotrimer and Nup54 could replace one of the chains of Nup62 to form a more stable complex. Although underlying mechanism of the dissociation of Nup62 homotrimer chains and replacement with other interacting partners is yet to be understood, we know that the formation of heterotrimeric assemblies (with Nup54) is an energetically favored reaction. It was shown earlier that the central channel and intermediate regions that connect the central channel to the NPC symmetric core undergo structural changes when frequency of nucleo-cytoplasmic transport is inhibited50. These structural changes in the NPCs may be the result of various rearrangement of complexes formed by the central channel Nups interacting via their coiled-coil regions.


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A very recent study showed that the levels of Nup62 in Drosophila embryos is significantly higher in nuclear envelope and cytosol as compared to Nup54 and Nup5816. This implies that the NPC contains higher levels of Nup62 either in self oligomerized or in complexes other than Nup62•Nup54•Nup58 or Nup62•Nup88•Nup214, suggesting that previously reported diverse stoichiometries may exist in the NPC and may alter upon changes in physiological processes17, 18, 27, 28. Nup62 is known to interact directly with Exo70, a member of the exocyst complex27. Our data confirmed this interaction and further we identified the minimal region required for these

interactions

(Figure

heterotrimeric model and

5).

Superimpositions

of

Nup62(362-425)•Exo70(33-81)

rat Nup62(362-425) homotrimeric structure suggests similar

interface in these two assemblies. Based on these observations, we propose that one chain of Nup62 homotrimer can be replaced by Exo70 α-helix to form a functionally different complex. The functional relevance of such complex is yet to be elucidated. In case of Encephalomyocarditis viral infection, the leader protein from virus causes hyperphosphorylation of Nup62 along with Nup214 and Nup153, leading to reduced localization of Nup62 to the NPC as well as nuclear trafficking30. Another study showed that, under chronic stress conditions, phosphorylation of human Nup62 at the Y422 residue causes accumulation of Nup62 protein pool in the cytoplasm31. We propose that the cytosolic Nup62 in these cases can be in the form of a homodimer and/or homotrimer. However, whether the cytosolic form of Nup62 is present as a homo-oligomer alone or in conjugation with some other factors is yet to be unveiled. In summary, coiled-coil motif present in Nup62 imparts ability to form a homotrimer as well as heterotrimers with either Nup54 or Nup54•Nup58. The capability of Nup62 to form different complexes demonstrate that how the coiled-coil motif contributes in the modular assemblies of various triple helix bundles.


Biochemistry

26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACKNOWLEDGMENTS We thank Dr. Saikrishnan Kayarat, IISER-Pune for the X-ray diffraction facility for initial diffraction screening of crystals. We thank Dr. Nicola Dimitri and Dr. Maurizio Polentarutti (Elettra Synchrotron) for their help at the XRD1 beamline. We thank Dr. Shekhar Mande and Dr Janesh Kumar and members of structural biology group from NCCS for comments on the manuscript. Plasmid having rat Exo70 cDNA was kind gift from Dr. Jomon Joseph Laboratory, NCCS Pune. AUTHOR CONTRIBUTIONS The study was designed by R.C. and experimental contributions were made by P.S.D, A.R.C and R.C. The manuscript was written by P.S.D., P.J.S. and R.C. FUNDING SOURCE STATEMENT This work is supported by Science and Engineering Research board (SERB) grants (RC/BB/SO/030/2013 & SR/S2/RJN-48/2012) and NCCS intra-mural funding to R.C., who is a Ramanujan Fellow. P.S.D. is supported by University Grants Commission (UGC), New Delhi, India. P.J.S. is supported in grant: RC/BB/SO/030/2013 and A.R.C was supported in grant: SR/S2/RJN-48/2012.

SUPPORTING INFORMATION PARAGRAPH Coiled-coil domains of channel Nups (Figure S1); Superimposition of rat Nup62(362-425) homotrimer on rat Nup62(362-425)•Nup54(346-407) heterotrimer (Figure S2); Size exclusion chromatography coupled Multi-angle Light Scattering (SEC-MALS) analysis (Figure S3); Superimposition of rat Nup62(362-425) homotrimer on Xenopus CTC sub-complex (Figure S4); Helical wheel representation of the coiled-coil sequence for rat


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Biochemistry

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Nup62(362-425)•Nup54(346-407) heterotrimer and Xenopus CTC sub-complex (Figure S5); Modelling of rat Exo70(33-81) coiled-coil region with rat Nup62(362-425) (Figure S6); Sequences of GST-fusions of Exo70 proteins used in this study (Figure S7). Table S1: PISA analysis indicating salt bridges in various Nup62 complexes Table S2: PISA analysis showing complex formation score and the effective solvation energies for complexes of Nup62.

STRUCTURAL DATA The coordinate file and the experimental data have been submitted at worldwide Protein Data Bank. PDB Id for the crystal structure of rat Nup62(362-425) is 5H1X. The coordinates will be released immediately upon publication.


Biochemistry

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