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 ␤-barrel with a PWWP-specific long loop connecting ␤2 and ␤3 (PR-loop), followed by a helical region including two ␣-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the ␤1/␤2 loop (␤-␤ arch) and the ␤3/␤4 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.

Methods for quantitative oral administration of various substances to Caenorhabditis elegans are needed. Previously, we succeeded in oral administration of hydrophilic substances using liposomes. However, an adequate system for delivery of hydrophobic chemicals was not available. In this study, we developed a method for oral administration of lipid-soluble substances to C. elegans. γ-cyclodextrin (γCD), which delivers hydrophobic chemicals, was used to make micro-particles of inclusion compounds that can be ingested by bacteriophagous nematodes, which do not distinguish these micro-particles from their food bacteria. Successful oral delivery of the hydrophobic fluorescent reagent 3,3'-dioctadecyloxacarbocyanine perchlorate into the intestines of C. elegans was observed. Oral administration of the hydrophobic antioxidants tocotrienol, astaxanthin, or γ-tocopherol, prolonged the nematode lifespan; tocotrienol rendered them resistant to infection with the opportunistic pathogen Legionella pneumophila. In contrast, older conventional delivery methods that involve incorporation of chemicals into the nematode growth medium or pouring chemicals onto the plate produced weaker fluorescence and no longevity effects. Our method efficiently and quantitatively delivers hydrophobic solutes to nematodes, and a minimum effective dose was estimated. In combination with our liposome method, this γCD method expands the usefulness of C. elegans for the discovery of functional food factors and for screening drug candidates.

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a b-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 3 10 2 M À1 . We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spinrelaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra a-helix was predicted. An NMR structural determination confirmed the formation of an a-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentrationdependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal a-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.Keywords: WW domain; dimerization; stability; dynamics; ultracentrifuge; NMR The WW domain, which consists of about 40 amino acid residues, is a monomeric protein-binding module with a three-stranded antiparallel b-sheet motif. The name WW is derived from the presence of two highly conserved tryptophan residues with 20-23 amino acid spacing in the polypeptide sequence. They are located on opposite faces of the twisted b-sheet, and the N-terminal tryptophan residue contacts the highly conserved proline residue Reprint requests to: Shigeyuki Yokoyama, Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; e-mail: yokoyama@biochem.s.u-tokyo.ac.jp; fax: 81-45-503-9195.Abbreviations: SAV1 WW2, the second WW domain of Salvador protein 1; MAGI1 WW2, the second WW domain of membrane associated guanylate kinase, WW and PDZ domain containing 1.Article and publication are at http://www.proteinscience.org/cgi

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