Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element.
The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.
Baburajendran
Nithya
N
Genome Institute of Singapore, Singapore.
Palasingam
Paaventhan
P
Ng
Calista Keow Leng
CK
Jauch
Ralf
R
Kolatkar
Prasanna R
PR
D000818:Animals; D001483:Base Sequence; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism; D055029:Inverted Repeat Sequences; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051898:Smad1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
D019485:Bone Morphogenetic Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D004247:DNA