Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Abstract

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

Figure 1.

(A) Sequence alignment of the N-terminal regions of the HDGF family members. Accession codes used are as follows: in Mus musculus, HRP-3, NP_038914; HDGF, BAA09838; HRP-2, NP_032259; LEDGF, NP_598709; HRP-1, NP_032258; in Bos taurus, HRP-4, CAB40348. Gray-colored residues indicate that more than half of the six residues in the same position are identical [the following residues in parentheses are grouped into the same type: (I, L, M, V) and (E, D)]. The PWWP motif is indicated by a black background. (B) Alignment of the Pfam PWWP domains (Bateman et al. 2004) shown in the HMM-Logo format, where the tallest residues are invariant (Schuster-Böckler et al. 2004). The sequence of the PWWP domain of HRP-3 is shown below with the secondary structural elements colored. Circles and asterisks indicate highly conserved residues involved in the interactions between α3 and the β-barrel substructure, and the formation of the cavity, respectively, as described in the text.

Figure 2.

(A) Stereo view illustrating a trace of the backbone atoms for the ensemble of the 20 lowest energy structures of the mouse HRP-3 PWWP domain (residues 8–90). (B) Ribbon diagrams of the PWWP domains from the mouse HRP-3, the S. pombe protein SPBC215.07c (PDB code 1H3Z), and the mouse DNA methyltransferase Dnmt3b (1KHC) in the same view. Four residues in parentheses indicate those of the PWWP motif of each PWWP domain. The α-helices, β-strands and the PR-loop are colored pink, green, and dark orange, respectively.

Figure 3.

(A) Stereo view illustrating the interactions between α3 and the β-barrel substructure. β1, β2 and the connecting loop (β-β arch) are depicted in green, while α3 is pink. Shown are the PHWP residues (hot pink), Phe49 and the β3/β4 loop (brown), and Phe81 (navy). A hydrogen bond is depicted by a red dotted line. (B) Comparison of the second residue of the PWWP motif between the HRP-3 and S. pombe domains. β1, β2 and β5 are depicted in green. van der Waals surfaces are shown for the side chain atoms of His24 (hot pink) and Phe63 (navy) in the HRP-3 domain, and for those of Trp138 (hot pink) and Thr184 (navy) in the S. pombe domain. Residues in the β-β arch are colored orange.

Figure 4.

Topological comparisons of the PWWP and Tudor domains. Note that the region depicted by a broken line region corresponds to a 3₁₀-helix in the two domains. An asterisk indicates a β-bulge.

Figure 5.

(A) Binding sites of the Tudor (PDB code 1G5V), MBT (1OZ3) and chromo (1KNA) domains. In the MBT domain, the region encompassing residues 349–44 is shown. The equivalents to β1 and β2 in the PWWP domain are colored blue, while those corresponding to β3 and β4 are dark green. Aromatic residues and negatively charged residues involved in ligand binding are colored dark orange and red, respectively. (B) A cavity as a putative binding site in the PWWP domain (left) and molecular surface representations showing hydrophobic residues in green (right). The figure on the right was prepared with GRASP (Nicholls et al. 1991). Residues involved in cavity formation are shown. Arrows indicate the cavity as a putative binding site. The red hatched circle indicates the position corresponding to that in which a missense mutation causes ICF syndrome in the human Dnmt3b PWWP domain, as described in the text. (C) Molecular surface representations of the electrostatic potential (blue, positive; red, negative) of the PWWP domain, calculated by GRASP. The middle view is in the same orientation as those in (B). On the left and right, the views are rotated by 90° around the vertical axis.