Human YTH domain family, member 1, YTH domain, in complex with N(6)-methyladenosine RNA

Target ID:

YTHDF1

Deposition Date:

Tuesday 16th September 2014

Families:

Miscellaneous

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4RCJ

Authors:

Tempel W, Xu C, Bountra C, Arrowsmith CH, Edwards AM, Min J

Site:

Toronto

4RCJ

tabs

Structure Details

We determined the crystal structure of YTHDF1 (aa 365-554) in complex with 5-mer m6A RNA at a resolution of 1.60 Ã… (Table S1). In the complex structure, nucleotides 5'-GG(m6A) exhibited density for both the backbone and nucleobases. Additional density was interpreted as representing nucleotide (C+1) and the phosphate of (U+2)-3'. The m6A RNA bound to a positively charged concave of YTHDF1 in an extended confirmation. The m6A binding pocket of the YTHDF1 YTH domain was similar to that of the reported YTHDC1 YTH domain, and was composed of the C-termini of β1, α1, β2, the N-terminus of α2, and the loop between β4 and β5 (named L45). Specifically, the m6A was accommodated in a pocket composed of Trp411, Trp465 and Trp470, with the ring planes of Trp411 and Trp470 parallel to each other and perpendicular to the ring plane of Trp465. The N6-methyl bond was coplanar with the m6A purine ring, with the methyl group pointing toward Trp465 and the m6A adenine moiety sandwiched by the rings of Trp411and Trp4. The CH-π interaction between the N6 methyl moiety and the aromatic cage, together with the π-π interactions between the adenine base and the aromatic residues, constituted the basis of the m6A specific recognition. Additionally, the m6A adenine formed hydrogen bonds with the YTHDF1 YTH domain, including the N3 of m6A and the backbone NH of Tyr397, and the N6 of m6A and the backbone carbonyl of Cys412. The side chain oxygen of Asp401 was located in close proximity of the N1 of m6A.

Materials and Methods

YTHDF1

PDB:4RCJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_060268.2
Entry Clone Source:MGC AT61-H6 (NP_060268.2)
SGC Clone Accession:YTHDF1_7 (JMC02A:E07): 365-554
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:BL21 (DE3) Codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGHPVLEKLKAAHSYNPKEFEWNLKSGRVFIIKSYSEDDIHRSIKYSIWCSTEHGNKRLDSAFRCMSSKGPVYLLFSVNGSGHFCGVAEMKSPVDYGTSAGVWSQDKWKGKFDVQWIFVKDVPNNQLRHIRLENNDNKPVTNSRDTQEVPLEKAKQVLKIISSYKHTTSIFDDFAHYEKRQEEEEVVRKERQ
Vector:pET28-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 ºC. 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 ºC. The temperature of the media was reduced to 15 ºC 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 ºC, and cell pellets collected by centrifugation and frozen at -80 ºC.

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 10,000 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 YTHDF1 YTH domain (10 mg /ml) was mixed with the 5-mer RNA GG(m6A)CU in a ratio of 1:2 and put on ice for 30 minutes before crystallization. The complex was crystallized in a buffer containing 25% PEG-3350, 0.2 M sodium chloride, 0.1 M bis-tris, pH 6.5 .The crystals were soaked in a cryoprotectant consisting of 85 to 90% reservoir solution and 10 to 15% glycerol (v/v).
NMR Spectroscopy:
Data Collection:
Data Processing: