Human regulation of nuclear pre-mRNA domain containing 1A, CTK3 domain, in complex with peptide

Target ID:

RPRD1A

Deposition Date:

Thursday 28th March 2013

Families:

Miscellaneous

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

4JXT

Authors:

Ni Z, Xu C, Tempel W, El Bakkouri M, Loppnau P, Guo X, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Min J, Greenblatt JF

Site:

Toronto

4JXT

tabs

Structure Details

The RPRD1A CID contained eight α-helices arranged in a right-handed superhelical manner. A concave channel accommodated the CTD decapeptide PSYSPTSPSY (S7P underlined) in a linear conformation such that two S7P residues, S7aP and S7bP, occupied the channel entrance and exit, respectively. We observed many CID-CTD contacts. CID N18 formed water-mediated hydrogen bonds with the S7a phosphate group and the backbone carbonyl group of Y1b and further hydrogen bonds with the backbone amide of Y1b. N64 and D65 hydrogen-bonded to the Y1b phenolic hydroxyl group deeply buried inside the center of the channel. N64 also contacted the P3b carbonyl group via a hydrogen bond. Q20 formed hydrogen bonds with the P6a carbonyl group, the CTD backbone amide of S2b and the S5b hydroxyl group. R114 hydrogen-bonded with the carbonyl groups of both T4b and P6b. N69 formed water-mediated hydrogen bonds with both the backbone amide and phosphate group of S7b. In addition, I110 contacted both P3b and T4b through hydrophobic interactions. Finally, the two S7P phosphate groups pointed toward nearby positively charged areas of the CID surface, one of them close to R72. Altogether, direct and indirect hydrogen bonds, hydrophobic interactions and electrostatic forces contribute to the interaction network between the S7P CTD and the RPRD1A CID.

Materials and Methods

RPRD1A

PDB:4JXT

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_060640.2.
Entry Clone Source:MGC DNA 05-F4 (NP_060640.2.)
SGC Clone Accession:RPRD1A (DCC020G05): 2-137
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:BL21 (DE3) Codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQG SAGALESSLDRKFQSVTNTMESIQGLSSWCIENKKHHSTIVYHWMKWLRRSAYPHRLNLFYLANDVIQNCKRKNAIIFRESFADVLPEAAALVKDPSVSKSVERIFKIWEDRNVYPEEMIVALREALSTTF
Vector:pET15-MHL

Growth

Medium:
Antibiotics:
Procedure:A 250 mL flask containing LB (Sigma L7658) supplemented with 50 µg/mL kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 °C. Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 mL of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ mL kanamycin and 600 µl antifoam 204 (Sigma A-8311) was inoculated with 50 mL overnight LB culture, and incubated at 37 °C. The temperature of the media was reduced to 15 °C one hour prior to induction and induced at OD600 = 6 with 100 µM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 °C, and cell pellets collected by centrifugation and frozen at -80 °C.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 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. pTEV was added to eluted protein at 1:20 for eluted protein and dialyze against gel filtration buffer overnight to remove His-tag.

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 2L 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 25 mg/mL.
Ligand
MassSpec:
Crystallization:The RPRD1A CID domain was mixed with phosphorylated Ser5 CTD in a ratio of 1:3 and put on ice for 30 minutes before crystallization. The RPRD1A complex was crystallized in a buffer containing 10.0 % Iprop, 20.0% PEG 4K, 0.1 M Hepes ph7.5. The crystals were soaked in a cryoprotectant consisting of 88% reservoir solution and 12% glycerol (v/v).
NMR Spectroscopy:
Data Collection:
Data Processing: