Structural basis of binding of P-body-associated proteins GW182 and ataxin-2 by the Mlle domain of poly(A)-binding protein

Abstract

Poly(A)-binding protein (PABPC1) is involved in multiple aspects of mRNA processing and translation. It is a component of RNA stress granules and binds the RNA-induced silencing complex to promote degradation of silenced mRNAs. Here, we report the crystal structures of the C-terminal Mlle (or PABC) domain in complex with peptides from GW182 (TNRC6C) and Ataxin-2. The structures reveal overlapping binding sites but with unexpected diversity in the peptide conformation and residues involved in binding. The mutagenesis and binding studies show low to submicromolar binding affinity with overlapping but distinct specificity determinants. These results rationalize the role of the Mlle domain of PABPC1 in microRNA-mediated mRNA deadenylation and suggest a more general function in the assembly of cytoplasmic RNA granules.

FIGURE 1.

Crystal structure of the PABPC1 Mlle domain complexed with the DUF region of GW182. a, schematic representation (left, purple) and surface (right) of the Mlle domain with the bound GW182 peptide (cyan). The Mlle domain forms a five-helix bundle with the peptide sitting in a shallow hydrophobic pocket formed by helices α2 and α3. The GW182 peptide N and C termini sit together on the same side of the Mlle domain. b, intra- and intermolecular hydrogen bonds in the GW182 peptide. Bound water molecules (blue circles) form a network of hydrogen bonds (dash lines) above the side chain of Phe¹³⁸⁹. The GW182 peptide forms two β-turns, which position the hydrophobic residues Phe¹³⁸⁹ and Trp¹³⁹⁵ into a shallow groove between Mlle helices α2 (shown diagonally on the left) and α3. Intermolecular hydrogen bonds between Phe¹³⁸⁹ and Mlle Gly⁵⁷⁹ and between Gly¹³⁹² and Mlle Glu⁵⁶⁴ are shown. For clarity, only GW182 peptide residues are labeled. c, hydrogen bonding between the C-terminal portion of the GW182 peptide and Mlle helix α2. d, hydrophobic residues in the GW182 peptide responsible for binding to the Mlle domain. The peptide is shown flipped 180° horizontally relative to b to display the surface facing the Mlle domain. The surface is color-coded by element type to highlight the nonpolar surface centered around the aromatic residues Phe¹³⁸⁹ and Trp¹³⁹⁵.

FIGURE 2.

Solution studies of PABPC1 Mlle binding the GW182 DUF region. a, ITC trace (top) and integrated areas (bottom) for GW182-(1380–1401) binding to the PABPC1 Mlle domain. b, backbone amide ¹⁵N{¹H} heteronuclear nuclear Overhauser effects for ¹⁵N-labeled GW182 peptide bound to Mlle domain. Values above 0.6 indicate highly ordered residues. c, chemical shift changes in the ¹⁵N-labeled PABPC1 Mlle domain (residues 544–626) upon addition of a peptide comprising the GW182 DUF region. Shifts are calculated as a weighted average in ppm as (ΔH² + (ΔN/5)²)^1/2. d, mapping of the chemical shift changes by residue color (white, no change; black, maximum change) onto a schematic representation of the unliganded Mlle domain. For reference, the position of the GW182 peptide in the x-ray crystal structure is shown.

FIGURE 3.

Crystal structure of the PABPC1 Mlle domain complexed with the PAM2 motif of Ataxin-2. a, schematic representation (left, purple) and surface (right) of the Mlle domain with bound Ataxin-2 peptide (cyan). The peptide wraps around the Mlle domain, binding to helices α2, α3, and α5 with its N and C termini on opposite faces. b, details of the N-terminal portion of Ataxin-2. For clarity, residues in Ataxin-2 are underlined. L914 binds the hydrophobic pocket formed by Leu⁵⁸⁵, Ala⁶¹⁰, and the aliphatic parts of Lys⁶⁰⁶ and Glu⁶⁰⁹. P916 inserts into a small pocket formed by the side chains of Met⁵⁸⁴, Val⁶¹³, Leu⁶¹⁴, and His⁶¹⁷. A918 makes a hydrophobic contact with the base of the Met⁵⁸⁴ side chain. The side chain of Lys⁵⁸⁰ forms intermolecular hydrogen bonds with carbonyls of P916 and A918. The β-turn in the peptide is stabilized by a pair of hydrogen bonds between N915 and the backbone amides of N917 and A918. c, in the C-terminal portion, F921 is a key hydrophobic residue and binds the shallow pocket in Mlle formed by Thr⁵⁸², Phe⁵⁶⁷, Leu⁵⁸⁶, and Gly⁵⁶³. The backbone of F921 is rigidified by hydrogen bonds involving Gly⁵⁷⁹, Gln⁵⁶⁰, and N922 and a bound water molecule.

FIGURE 4.

Comparison of the Mlle complexes with the DUF region of GW182 and the PAM2 motif of Ataxin-2. The peptides from GW182 (magenta) and Ataxin-2 (cyan) follow distinctly different courses across the face of the Mlle domain (blue) but overlap in the core zone around residues Phe¹³⁸⁹/Phe⁹²¹. The phenylalanine rings of the two residues superpose to within 0.5 Å. Additional hydrophobic contacts are provided by Trp¹³⁹⁵ of GW182 and by Leu⁹¹⁴ of Ataxin-2.

FIGURE 5.

Sequence conservation of the Mlle-PAM2 binding determinants and model of PABPC1 on the mRNA poly(A) tail. a, DUF sequence of GW182 aligned against PAM2 sequences from Ataxin-2, eukaryotic release factor 3b (eRF3), Tob1 (transducer of Erb1), poly(A)-specific ribonuclease 3 (PAN3), and ubiquitin-specific peptidase 10 (USP10). The most highly conserved residues are underlined. b, sequence alignment of the Mlle domains from human UBR5 and PABPC1. The secondary structure is shown labeled according to Mlle from PABPC1. The domain from UBR5 lacks helix α1. The MLLE motif found in all Mlle domains is underlined. c, model of PABPC1 with Mlle-interacting proteins. Typical mRNA poly(A) tails will accommodate multiple PABPC1 proteins whose Mlle domains bind GW182 and PAM2-containing proteins.