Human lysine methyltransferase 2C, 6th PHD domain, in complex with H4 peptide

Target ID:

MLL3

Deposition Date:

Saturday 29th September 2018

Families:

Methyl-lysine readers

Related Diseases:

Cancer and epigenetics

Structure type:

protein-protein

Download Coordinates:

6MLC

Authors:

Dong A, Liu Y, Qin S, Lei M, Bountra C, Arrowsmith CH, Edwards AM, Min J

Site:

Toronto

tabs

Structure Details

The crystal structure of the MLL3 ePHD₆ (extended PHD₆, ePHD₆) domain revealed that it contains a canonical PHD domain preceded by a zinc knuckle composed of two antiparallel b strands and an a helix (b1: V1055-C1057, b2: G1061-S1064, a1: A1079-S1085). The MLL3 ePHD₆ domain formed a dimer through two N-terminal cysteine residues, which coordinated a zinc ion (C1058, C1060, Zn0) with two cysteine residues from each protomer of the dimer. In addition, a domain swapping between these two ePHD₆ molecules also contributed to the dimer formation, in which a zinc ion (H1059, C1069, C1079, C1081, Zn1) was chelated by three cysteine residues from one molecule and one histidine residue from the other molecule. The dimer involved only the preceding zinc fingers, but not the PHD domain itself. However, our gel filtration profiles showed that the extended PHD₆ domain of MLL3 behaved as a monomer in solution, like the PHD₆ domain itself. Therefore, the dimer was formed probably due to artificial crystal packing, which has been observed in other PHD proteins.

In the complex structure, the histone H4 peptide bound to one molecule of the two ePHD₆ molecules and was induced to form a β strand antiparalleling with β3 (I1100-C1103) of ePHD₆. The residues ₁₄GAKRHR₁₉ of the histone H4 peptide could be traced reliably. The H4G14, H4K16 and H4H18 residues formed main chain hydrogen bonds with the main chains of Q1102, I1100, E1097 and D1098 of MLL3, respectively, allowing the β-sheet formation between the H4 peptide and ePHD₆ of MLL3. The side chain of H4A15 was accommodated in a shallow hydrophobic pocket, formed by L1101, V1123 and A1127 of MLL3, while its main chain was restricted by W1109 of MLL3. The aliphatic chain of H4K16 stacked with the aromatic ring structure of W1109 and interacted with the hydrophobic side chain of I1100. H4R17 formed a salt bridge with E1120 and H4R19 formed a salt bridge with E1097. In addition to the salt bridge with E1120, H4R17 also formed a hydrogen bond with the main chain of E1097. Thus the positive side chain of H4R17 was clasped in a negative channel formed by E1120 and E1097. The imidazole ring of H4H18 lied against the side chain of Y1094 and was further restricted by the main chain carbonyl groups of L1084 from the preceding zinc finger (Zn1, V1055-S1085) and R1095 of PHD₆. The α-helix α1 from the zinc finger Zn1, which packed against the β4 (D1107-H1111) of ePHD₆, contributed to the formation of the peptide binding groove.

Materials and Methods

PHD6 domain of MLL3 in complex with histone H4

6MLC

NP_733751.2

32-F1

MLL3_ZNPHD6_GST3 (JMC076:C09): V1055-N1144

N-terminal tag:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD

KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPMLKMFEDRLCHKTYLNGDHVTHPD

FMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSSMGSSHHHHHHSSGLVPRGS

VWCRHCGATSAGLRCEWQNNYTQCAPCASLSSCPVCYRNYREEDLILQCRQCDRWMHAVCQNLNTEEEVENVADIGFDCSMCRPYMPASN

pET28GST-lic

Escherichiacoli BL21 (DE3)

Lex system, 37 °C 3-4h, 15 °C 18-24h

Lysis buffer: 20 mM Tris pH 7.5 (R.T.), 500 mM NaCl

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 200 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 seconds pulse at 80% of maximal frequency (8.0), 10 seconds rest, for 10 minutes total sonication time per pellet.

Wash buffer: 20 mM Tris pH 7.5, 1 M NaCl, 25 mM imidazole
Elution buffer: 20 mM Tris pH 7.5, 250 mM NaCl, 250 mM imidazole
Gel filtration buffer: 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT, 50 μM ZnCl₂

IMAC: Unclarified lysate was mixed with 3-4 mL of Ni-NTA superflow Resin (Qiagen) per 200 mL lysate. The mixture was incubated with mixing for at least 30 minutes at 4°C. The mixture was then loaded onto an empty comLum (BioRad) and washed with 40 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 16x60 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 concentrate sample (approx. 3 mL, concentrated by using 15 mL concentrators with a 3,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm) from the IMAC step was loaded onto the column at 1 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.

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 5 mg/mL.

Recombinant human MLL3 ePHD₆ domain was mixed with histone H4_1-20 peptide at molar ratio of 1:3 and co-crystallized using the sitting drop vapour diffusion method at 18 °C. The crystals were obtained in a buffer containing 2 M Na-Form, 0.1 M Tris, pH 8.5. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.