The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14

Abstract

MRG15 is a transcription factor expressed in a variety of human tissues, and its orthologs have been found in many other eukaryotes which constitute the MRG protein family. It plays a vital role in embryonic development and cell proliferation, and is involved in cellular senescence. The C-terminal part of MRG15 forms a conserved MRG domain which is involved in interactions with the tumor suppressor protein retinoblastoma and a nucleoprotein PAM14 during transcriptional regulation. We report here the characterization of the interaction between the MRG domain of human MRG15 and PAM14 using both yeast two-hybrid and in vitro binding assays based on the crystal structure of the MRG domain. The MRG domain is predominantly hydrophobic, and consists of mainly alpha-helices that are arranged in a three-layer sandwich topology. The hydrophobic core is stabilized by interactions among a number of conserved hydrophobic residues. The molecular surface is largely hydrophobic, but contains a few hydrophilic patches. Structure-based site-directed mutagenesis studies identified key residues involved in the binding of PAM14. Structural and biochemical data together demonstrate that the PAM14 binding site is consisted of residues Ile160, Leu168, Val169, Trp172, Tyr235, Val268, and Arg269 of MRG15, which form a shallow hydrophobic pocket to interact with the N-terminal 50 residues of PAM14 through primarily hydrophobic interactions. These results provide the molecular basis for the interaction between the MRG domain and PAM14, and reveal insights into the potential biological function of MRG15 in transcription regulation and chromatin remodeling.

Figure 1.

Structure of MRG15C. (A) A representative SIGMMA-weighted 2F _o–F _c map (1σ contour level) in the C-terminal region of one MRG15C subunit at 2.2 Å resolution. The final coordinates of the structure are shown as a ball-and-stick model. (B) Ribbon diagrams showing the overall structure of MRG15C in monomer (left panel) and homodimer (right panel). The disordered region connecting helices α2 and α3 is indicated with dashed lines. The conserved residues involved in formation of the hydrophobic dimer interface are shown with side chains. The PAM14 binding site and a hydrophilic patch near the C-terminal region are indicated by circles. (C) Secondary structure topology of MRG15C. α-Helices are shown as cylinders; β-strands as arrows. (D) Sequence comparison of the MRG domains between human MRG15 and its homologs. Hs_MRG15, human MRG15; MmMRG15, Mus musculus MRG15; Dm_MRG15, Drosophila melanogaster MRG15; Sp_ALP13, Schizosaccharomyces pombe altered polarity protein 13; Sc_EAF3p, Saccharomyces cerevisiae Esa1p associated factor 3 protein; At_MRG15, Arabidopsis thaliana MRG15; and Ce_MRG15, Caenorhabditis elegans MRG15. Strictly conserved residues are highlighted in shaded red boxes and conserved residues in open red boxes. The secondary structure of the MRG domain of human MRG15 is placed on top of the alignment.

Figure 2.

Yeast two-hybrid assay of the interaction between MRG15 and PAM14. MRG15, MRG15N, MRG15C, and MRG15C2 were cotransformed with pB42AD as the baits, and PAM14 and its fragments (PAM14N, PAM14M, and PAM14C) were cotransformed with pGilda as the preys. The pSH17-4 plasmid was used as the positive control and the pRFHM1 plasmid as the negative control for transcriptional activation. (A) Yeast two-hybrid analyses of pGilda-MRG15s (MRG15C and MRG15) with the empty prey plasmid pB42AD and pB42AD-PAM14s (PAM14N, PAM14M, PAM14C, and PAM14) with the empty bait plasmid pGilda. The baits or preys alone cannot activate the reporter markers by themselves. (B) Yeast two-hybrid analyses to determine the interactions of MRG15, MRG15N, MRG15C, and MRG15C2 with PAM14 and its fragments (PAM14N, PAM14M, and PAM14C). (Left to right) Full-length MRG15 (residues 1–323), MRG15N (residues 1–150), MRG15C (residues 151–323), and MRG15C2 (residues 173–323). Both MRG15 and MRG15C have direct interactions with PAM14 and PAM14N at comparable levels, whereas MRG15N and MRG15C2 have no interaction with PAM14 or its fragments.

Figure 3

In vitro binding assay of the interaction between MRG15C and PAM14. GST was used as the negative control, which cannot bind to MRG15C. (A) In vitro binding assays of MRG15C with GST-fused PAM14 (residues 1–127) and its three fragments GST-PAM14N (residues 1–50), GST-PAM14M (residues 49–84), and GST-PAM14C (residues 80–127). The results show that MRG15C has interactions with PAM14 and PAM14N, but no interaction with PAM14M and PAM14C. (B) In vitro binding assays of GST-PAM14N with wild-type MRG15C and representative MRG15C mutants containing mutations of positively-charged residues. Shown here are double mutations K159A/K165A, K159A/R269A, K159A/K296A, and N289A/K296A, and single mutation R204A. The results show that these mutations have no obvious effect on the binding affinity of MRG15C with PAM14N compared to wild-type MRG15C. (C) In vitro binding assays of GST-PAM14N with wild-type MRG15C and representative MRG15C mutants containing mutations of both hydrophobic and hydrophilic residues. Shown here are single mutations V169A, V169E, W172A, Q178A, Y183A, D208A, M228D, Y235A, R239A, R320A, I160A, I160D, L168A, N215A, N215D, V268A, V268D, and R269E. The results show that mutation L168A and V169A significantly reduce the binding affinity of MRG15C with PAM14N; mutation I160A, I160D, V169E, W172A, Y235A, V268A, V268D, and R269E completely abolish the binding of PAM14N; and other mutations have no significant impact on the binding of PAM14N. (D) In vitro binding assay of GST-PAM14 with P185A mutant MRG15C. The result shows that mutation P185A substantially decreases the binding of MRG15C with PAM14. (E) In vitro binding assay of MRG15N and MRG15C. (Lane 1) His-tagged MRG15N; (lane 2) mixture of His-tagged MRG15N and GST-tagged MRG15C; (lane 3) mixture of His-tagged MRG15N and GST-tagged MRG15C after being washed three times with a washing buffer. The result shows that MRG15N and MRG15C have no direct interaction with each other.

Figure 4.

The PAM14 binding site. (A) Electrostatic surface of the dimeric MRG15C. MRG15 is highly hydrophobic but contains a few hydrophilic surface patches. The PAM14 binding site is located in a shallow hydrophobic pocket. A negatively-charged surface patch is located near the C-terminal region on one face of the molecule and several positively-charged patches on the opposite face. The residues forming the hydrophobic pocket and the hydrophilic surface patches are highlighted. (B) A stereoview of the structure of the PAM14 binding site. The PAM14 binding site consists of residues Ile160, Leu168, Val169, Trp172, Tyr235, Val268, and Arg269, which form a shallow hydrophobic pocket on one surface of the molecule. The hydrophobic residues are shown with side chains.