Implication of tubby proteins as transcription factors by structure- based functional analysis

Science

Volumn 286, Issue 5447, 1999, Pages 2119-2125

  Boggon, Titus J ^a   Shan, Wei Song ^b   Santagata, Sandro ^b   Myers, Samuel C ^a   Shapiro, Lawrence ^a  

^a NEW YORK STRUCTURAL BIOLOGY CENTER   (United States)

^b MOUNT SINAI SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

TRANSCRIPTION FACTOR;

ARTICLE; CRYSTAL STRUCTURE; DNA BINDING; DNA SEQUENCE; GENE MUTATION; HEARING LOSS; OBESITY; PHENOTYPE; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN STRUCTURE; RETINA DEGENERATION; STRUCTURE ACTIVITY RELATION; TOPOGRAPHY;

ALTERNATIVE SPLICING; AMINO ACID SEQUENCE; ANIMALS; CELL LINE; CELL NUCLEUS; CRYSTALLOGRAPHY, X-RAY; DNA; EYE PROTEINS; HUMANS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; PROTEIN CONFORMATION; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; PROTEINS; RECOMBINANT PROTEINS; SEQUENCE ALIGNMENT; TRANS-ACTIVATION (GENETICS); TRANSCRIPTION FACTORS;

EID: 0032787396     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5447.2119     Document Type: Article

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19. The tubby COOH-terminal domain was expressed as a fusion protein with glutathione S-transferase (GST) in the pGEX-2T expression vector. The fusion protein was purified by affinity chromatography on glutathione sepharose. The tubby COOH-terminal domain was released by proteolysis with thrombin and purified to homogeneity by ion exchange chromatography on mono-S followed by gel filtration on a Superdex-200 column. Analytical gel filtration shows that the protein exists in solution as a monomer.
20. 6-soaked crystal at beamline X4A of the National Synchrotron Light Source (NSLS). Crystals were flash-cooled to 100 K in stabilization buffer supplemented with 30% ethylene glycol Four data sets were collected around the selenium K absorption edge on the SeMet crystal. Data were processed and merged with the programs DENZO and SCALEPACK (33). The structure of COOH-terminal domain of tubby was determined by MAD phasing (11) of the SeMet crystal Strong peaks in the Bijvoet difference Patterson map from the iridium derivative allowed two sites to be found and initial phases to be generated by the single isomorphous replacement with anomalous scattering method. Difference Fourier maps using these phases were then calculated in X-PLOR [A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, CT, 1992)] and used to determine the positions of the five ordered selenium atoms. Phases were generated from the selenium data sets, using λ1 as the pseudo-native, to 3.0 Å with the program SHARP (34) and were improved by solvent flattening and phase extension to 2.8 Å with the program Solomon (35). An initial model of 221 residues was built into the resultant maps with the program O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Cystallogr. A47, 110 (1991)], rigid body refined, and refined against the high-resolution native data using a maximum likelihood target function with the program CNS [A. T. Brünger et al., Acta Crystallogr. D54, 905 (1998)]; 265 amino acid residues (19 of which with alternative conformations), 509 water molecules, and two phosphate ions were found and built into the structure.
21. 6-soaked crystal at beamline X4A of the National Synchrotron Light Source (NSLS). Crystals were flash-cooled to 100 K in stabilization buffer supplemented with 30% ethylene glycol Four data sets were collected around the selenium K absorption edge on the SeMet crystal. Data were processed and merged with the programs DENZO and SCALEPACK (33). The structure of COOH-terminal domain of tubby was determined by MAD phasing (11) of the SeMet crystal Strong peaks in the Bijvoet difference Patterson map from the iridium derivative allowed two sites to be found and initial phases to be generated by the single isomorphous replacement with anomalous scattering method. Difference Fourier maps using these phases were then calculated in X-PLOR [A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, CT, 1992)] and used to determine the positions of the five ordered selenium atoms. Phases were generated from the selenium data sets, using λ1 as the pseudo-native, to 3.0 Å with the program SHARP (34) and were improved by solvent flattening and phase extension to 2.8 Å with the program Solomon (35). An initial model of 221 residues was built into the resultant maps with the program O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Cystallogr. A47, 110 (1991)], rigid body refined, and refined against the high-resolution native data using a maximum likelihood target function with the program CNS [A. T. Brünger et al., Acta Crystallogr. D54, 905 (1998)]; 265 amino acid residues (19 of which with alternative conformations), 509 water molecules, and two phosphate ions were found and built into the structure.
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52. Ribbon diagram figures in this manuscript were made with the program SETOR [S. V. Evans J. Mol. Graphics 11, 134 (1993)], and the sequence alignment was made with DNASTAR [DNASTAR, Molecular Biotechnology, 5, 185 (1996)].
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55. We are grateful to C. Ogata and the staff of the NSLS beamline X4A for help with data collection. We thank W. A. Hendrickson, T. Harris, P. Kwong, and P. Scherer for many helpful discussions. T.J.B. is the recipient of a Wellcome Trust International Prize Travelling Research Fellowship (056509/Z/98/Z). L.S. is the recipient of a Career Scientist Award from the Irma T. Hirschl Foundation. This work was supported in part by a pilot study grant from Structural Genomix. Beamline X4A at the NSLS, a U.S. Department of Energy facility, is supported by the Howard Hughes Medical Institute. Coordinates have been deposited in the Protein Data Bank (accession code 1C8Z).