R ESEARCH A RTICLES resolution crystal structure by MAD analysis of the selenomethionyl (SeMet) protein (14) Implication of Tubby Proteins (Tables 1, 2, and 3). The structure reveals a striking fold in which a central hydrophobic as Transcription Factors by helix at the COOH-terminus wholly traverses the interior of a closed 12-stranded ␤ barrel Structure-Based Functional (Fig. 2). This arrangement is unique among known protein structures. The tubby ␤ barrel adopts an alternating Analysis up-down nearest-neighbor topology, such Titus J. Boggon,1 Wei-Song Shan,2 Sandro Santagata,3 that hydrogen bonding is in the antiparallel mode for all strands. We have numbered the Samuel C. Myers,1 Lawrence Shapiro1* strands of the barrel as 1 through 12 in se- quence order. Several excursions in the loops Tubby-like proteins (TULPs) are found in a broad range of multicellular organ- between these strands are observed. A three- isms. In mammals, genetic mutation of tubby or other TULPs can result in one stranded ␤ sheet intervenes in the 9 and 10 or more of three disease phenotypes: obesity (from which the name “tubby” connection, and we have thus designated is derived), retinal degeneration, and hearing loss. These disease phenotypes these strands as 9A, 9B, and 9C. Similarly, indicate a vital role for tubby proteins; however, no biochemical function has four helices, H4, H6A, H6B, and H8, are yet been ascribed to any member of this protein family. A structure-directed found in the corresponding loop regions be- approach was employed to investigate the biological function of these proteins. tween strands of the main barrel. The barrel is The crystal structure of the core domain from mouse tubby was determined at slightly oblong, with C␣ to C␣ widths across it a resolution of 1.9 angstroms. From primarily structural clues, experiments were varying between 18 and 22 Å. From top to devised, the results of which suggest that TULPs are a unique family of bipartite bottom, the barrel measures ϳ18 Å. The transcription factors. whole domain has maximum dimensions of 40 Å in the direction parallel to the central The tub gene was initially identified by posi- closely related to one another. These NH2- helix H12, and 51 Å in the perpendicular tional cloning of the genetic locus responsible terminal regions, however, are often similar plane. Helix H0 at the NH2-terminus caps the for the autosomal recessive obesity syndrome in cross-species orthologs. They comprise top of the barrel, and the long hydrophobic in the tubby strain of obese mice (1, 2). TubϪ/Ϫ about 180 to 280 amino acids in tubby and helix H12 traverses the inside of the barrel mice (3) also exhibit progressive sensorineural TULPs 1 through 3. from top to bottom. The possibility had pre- degeneration of the retina and cochlear hair Recent technological developments have viously been suggested that the H12 sequence cells, the primary sensory neurons involved in opened a new possibility for the application might constitute a transmembrane domain hearing (4). Similarly, mutation of the human of structural biology: to efficiently answer (9). TULP1 gene is the genetic origin of retinitis biological questions at an early stage of their Helix H12 forms integral contacts in al- pigmentosa type 14 (RP-14), which can pro- investigation, where questions of protein most every part of the hydrophobic core. The gressively lead to total blindness (5, 6). These function are more appropriately phrased as crystallographic temperature factors within sensory defects arise as the result of neuronal “What does it do?” rather than “How does it this region are, on average, the lowest in the cell death by apoptosis (4, 7, 8). The tubby do it?” (10). The revolution in genomics has structure (27.8 Å2 for H12 compared to 41.6 obesity syndrome may also arise from cell provided comprehensive lists of potential bi- Å2 for all other regions). It is highly unlikely death in the appetite control center of the hy- ological actors in numerous systems and or- that this helix could adopt other conforma- pothalamus, where the tubby protein is highly ganisms, and the technological revolution in tions outside of the core. The mutation in the expressed (5, 9), but this has not been demon- crystallography, particularly in multiwave- tubby mouse abolishes a splice donor site in strated experimentally. length anomalous diffraction (MAD) analysis the 3Ј exon, resulting in a deletion of the last Tubby is the founding member of a mul- (11), has enabled a quantum leap in the ease 44 amino acids, which are replaced with 20 tigene protein family. At least four tubby-like and rapidity of structure solution. Our knowl- intron-encoded amino acids (1, 2). Thus, in proteins (tubby and TULPs 1 through 3) are edge and understanding of biological struc- the tubby mouse, the entire hydrophobic core conserved among different species of mam- tures has expanded to the degree that visual- of this domain will be disrupted, and it is mals, and tubby-like proteins are also found izing the three-dimensional structure of a new therefore almost certain that no functional in other multicellular organisms (9), includ- protein can often point the way to biological protein can be produced in these animals. ing plants (1). They have not, however, been insights that would have been difficult to find The electrostatic surface of the protein, identified in single-celled organisms. These by other types of analysis (12). We have generated with the program GRASP (15), proteins feature a characteristic ϳ270–amino applied such a “structure-based functional shows two conspicuous features (Fig. 3). acid “tubby domain” at the COOH-terminus genomics” approach to tubby-like proteins. First, a groove of highly positive charge runs that does not exhibit homology to other This approach enabled us to efficiently iden- about one-half of the way around the barrel. known proteins. Most tubby proteins include tify the likely biochemical mechanism of this This groove is ϳ50 Å long, varies between ϳ ϳ NH2-terminal regions that, in general, are not protein family. 12 to 20 Å in width, and is up to 9Åin depth. There are surface contributions from Crystal Structure of the Tubby strands 5, 6, 7, 8, 9, and 10, and the groove is 1Structural Biology Program, Department of Physiol- Core Domain bordered at the top (near to the NH -terminal 2 2 ogy and Biophysics, Department of Biochemistry and As the first step in characterizing the biolog- end) by helix H8 and at the bottom by the Molecular Biology, 3Ruttenberg Cancer Center, Mount Sinai School of Medicine of New York University, New ical function of tubby, we expressed its large 7-8 loop and the three-stranded “extra” York, NY 10029, USA. COOH-terminal core domain (residues 243 9ABC sheet. The second notable feature is a *To whom correspondence should be addressed. E- through 505) (Fig. 1) in a recombinant ex- smaller, primarily convex, patch of negative mail: [email protected] pression system (13) and determined its high- charge found on the face opposite to the www.sciencemag.org SCIENCE VOL 286 10 DECEMBER 1999 2119 R ESEARCH A RTICLES center of the large groove. the immunostaining patterns were not ob- positions 39 and 302) are also conserved in Mapping TULP1 mutations (6, 16), iden- served in these experiments. Caenorhabditis elegans. No bipartite NLS tified from RP-14 patients, onto the tubby Mouse tubby has four sequences that fit are evident. Only one of the potential NLS is structure reveals a striking pattern. All sur- consensus patterns (19) for nuclear localiza- within the COOH-terminal domain. This re- face mutations [with one exception, de- tion signals (NLS). These are: K39KKR, gion (K302RKK) is found at the base of 56 123 302 ␤ scribed in (17)] cluster within a relatively P RSRRAR, P RKEKKG, and K RKK -strand 3, and the K302 side chain points into small region of the large positively charged (20). These potential NLS are mostly con- the core of the protein to make a structural groove that wraps the barrel. Some of these served throughout the mammalian tubby-like salt bridge with D499 (20). Thus, we think it is disease-causing mutants, including Arg240 3 proteins, and some (corresponding to those at unlikely that this corresponds to a functional Pro240 and Arg3783 His378 (human TULP1 numbering), convert positively charged side chains to neutral ones, suggesting an impor- tant biological function dependent on the maintenance of a positive surface. On the basis of the contiguous arrangement of these mutants, we postulated that this surface might form a protein or nucleic acid binding site. Nuclear Localization of Tubby Protein Although the tissue distributions of tubby and other TULPs have been investigated by Northern (RNA) blotting and in situ messen- ger RNA (mRNA) hybridization (5, 9), cell type and subcellular protein localization have not been reported. To address these ques- tions, we generated antibodies against the purified tubby COOH-terminal domain pro- tein and used these, as well as antibodies raised against a peptide from the NH2-termi- nal region, for immunocytochemical staining and protein immunoblotting experiments (18). First, we tested, with both of these methods, for protein expression in several cell lines and tissues including kidney cells, mouse fibroblasts (L cells), hippocampal neurons, astrocytes, and the Neuro-2A cell line. As expected, tubby is expressed primar- ily in cells of neural origin and at low or undetectable levels in the nonneural cells that we tested. The polyclonal antibody raised against the tubby COOH-terminal domain and the NH2-terminal peptide antibody yield similar cell staining patterns and protein im- munoblots. However, the COOH-terminal domain antibody gives protein immunoblots containing other bands near the tubby molec- ular weight.