X-ray structure of the human hyperplastic discs protein: an ortholog of the C-terminal domain of poly(A)-binding protein

Abstract

The poly(A)-binding protein (PABP) recognizes the 3' mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein-protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-A resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four alpha-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Figure 1

Domain organization of PABP and HYD protein families. (A) Sequence alignment of the conserved C-terminal domain of PABP (Homo sapiens, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Nicotiana tabacum), the orthologous region of the three HYD proteins (H. sapiens, Rat norvegicus, D. melanogaster), and a small ORF from the Arabidopsis thaliana genome. Sequence conservation among family members is color-coded by a yellow-green continuum; pale yellow represents low homology, dark green represents identity. (B) The four RRM domains of PABP (Top) are depicted by red boxes and are connected by interdomain linkers depicted as lines. The conserved C-terminal portion of the protein is shown as a blue ellipse. The location of the common coxsackie and poliovirus 2A protease site is indicated by the blue arrow. The HYD protein's domain organization (Middle) depicts the PABP-like region as a blue ellipse, and the HECT domain as a yellow hexagon. The PABP-like region of the A. thaliana small ORF (Bottom) is also represented as a blue ellipse.

Figure 2

Structure and surface properties of the amino (Left) and carboxyl (Right) faces of HYD-P and homology model of PABP-C. (A) ribbons (51) drawing of the HYD-P domain, where the four α-helices are labeled H1, H2, H3, and H4, as are the N and C termini. (B and C) grasp (52) representations of the chemical properties of the solvent-accessible molecular surface of HYD-P (B) and PABP-C (C) calculated by using a water probe (radius = 1.4 Å.). The surface electrostatic potential is color-coded red and blue, representing electrostatic potentials <−10 to >+10 kBT, where kB is the Boltzmann constant and T is the temperature. The calculations were performed with an ionic strength of 0 and dielectric constants of 80 and 2 for solvent and protein, respectively (53). (D and E) Depiction of the molecular surfaces of HYD-P (D) and PABP-C (E), color-coded green for phylogenetic conservation. Conserved amino-face residues of PABP-C are identified by a one-letter code.

Figure 3

The hydrophobic core of HYD-P. ribbons (51) stereodrawing of residues that constitute the hydrophobic core of the HYD-P domain. α-helices are labeled H1, H3, and H4 (see Fig. 2). Amino acids, labeled in gold, are identified by a one-letter code.

Figure 4

The PABP-C and HYD-P domains bind to Paip1 in vitro. Coomassie blue-stained SDS/PAGE gel of GST-affinity assays shows binding of Paip1 to both HYD-P (lane 2) and PABP-C (lane 3) with appropriate negative controls (lanes 1 and 4).