Human nucleoporin 43kDa

Target ID:

NUP43

Deposition Date:

Friday 30th November 2012

Families:

Miscellaneous

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

4I79

Authors:

Xu C, Tempel W, Li Z, He H, Wernimont AK, Bountra C, Arrowsmith CH, Edwards AM, Min J

Site:

Toronto

4I79

tabs

Structure Details

Human NUP43 adopts a canonical β-propeller fold, consisting of seven WD40 repeats (13-73, 77-116, 134-168, 175-212, 220-257, 264-344, 353-380 plus 5-8) and each WD40 repeat of NUP43 is composed of four anti-parallel β strands, resembling other WD40 containing proteins, such as WDR5, WDR61 and EED. There are two molecules of hNUP43 in the asymmetric unit and the global RMSD (Root Mean Square Deviation) between backbones of the two molecules is only 0.2 Å. Most of residues in the two molecules are visible. However, residues Met1-Glu3, Phe52-Phe61, Gly123-Y129, Thr293-Ser328, and Thr333-Asp338 are invisible in both molecules in the same asymmetric unit. In contrast, Gly50 and Asp51 are missing in only one molecule and Arg339 is missing in the other molecule. All of the missing residues lie either in the terminus or in the loop region, and they are invisible presumably due to the flexibility of these regions. Slight structural deviations come from flexible loop regions. The only exception comes from Glu62-Gln66, which include the very beginning of β1D. Probably these four residues also have internal flexibility because they mmediately follow the invisible loop Phe52-Phe61.

Materials and Methods

NUP43

PDB:4I79
Entry Clone Accession:NP_942590.1
Entry Clone Source:MGC AT85-H7 (BC065028)
SGC Clone Accession:NUP43_BV1 (JMC015:D03): M1-S380
Tag:N-terminal tag: MGSSHHHHHHSSGLVPLGS
Host:baculovirus
Vector:pFBOH-Lic

Sequence: MGSSHHHHHHSSGLVPRGSMEEIYAKFVSQKISKTRWRPLPPGSLQTAETFATGSWDNEENYISLWSIGDFGNLDSDGGFEGDHQLLCDIRHHGDVMDLQFFDQERIVAASSTGCVTVFLHHPNNQTLSVNQQWTTAHYHTGPGSPSYSSAPCTGVVCNNPEIVTVGEDGRINLFRADHKEAVRTIDNADSSTLHAVTFLRTPEILTVNSIGQLKIWDFRQQGNEPSQILSLTGDRVPLHCVDRHPNQQHVVATGGQDGMLSIWDVRQGTMPVSLLKAHEAEMWEVHFHPSNPEHLFTCSEDGSLWHWDASTDVPEKSSLFHQGGRSSTFLSHSISNQANVHQSVISSWLSTDPAKDRIEITSLLPSRSLSVNTLDVLGPCLVCGTDAEAIYVTRHLFS

Growth

Procedure:Baculovirus P1, P2, P3

Purification

Procedure:
IMAC: Unclarified lysate was mixed with 1-2 mL of Ni-NTA superflow Resin (Qiagen) per 40 mL lysate. The mixture was incubated with mixing for at least 45 minutes at 4oC. The mixture was then loaded onto an empty comLum (BioRad) and washed with 100 mL wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280

Gel filtration chromatography: An XK 26x65 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 1.0 mL/min. The dialyzed sample from the IMAC step (approx. 15 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure: Frozen cell pellet contained in bags (Beckman 369256) obtained from 6L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 20 mg/mL.

Structure Determination

Crystallization:Recombinant human NUP43 WD40 repeats was crystallized using the hanging dropvapour diffusion method at 18 °C. The crystals were obtained in a buffer containing 0.1 M Sodium Hepes, pH 7.5, 0.1 M Ammonium Sulphate, 25 % PEG 3350. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 12% glycerol.