ABSTRACT
Overexpression of multiple copies in T-cell lymphoma-1 (MCT-1) oncogene accompanies malignant phenotypic changes in human lymphoma cells. Specific disruption of MCT-1 results in reduced tumorigenesis, suggesting a potential for MCT-1-targeted therapeutic strategy. MCT-1 is known as a cap-binding protein and has a putative RNA-binding motif, the PUA-domain, at its C-terminus. We determined the crystal structure of apo MCT-1 at 1.7 Å resolution using the surface entropy reduction method. Notwithstanding limited sequence identity to its homologs, the C-terminus of MCT-1 adopted a typical PUA-domain fold that includes secondary structural elements essential for RNA recognition. The surface of the N-terminal domain contained positively charged patches that are predicted to contribute to RNA-binding.
Subject(s)
Cell Cycle Proteins/chemistry , Mutation , Oncogene Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Alanine/chemistry , Alanine/genetics , Amino Acid Motifs , Amino Acid Sequence , Biophysical Phenomena , Cell Cycle Proteins/genetics , Chromatography, Gel , Circular Dichroism , Cloning, Molecular , Crystallography/methods , DNA, Complementary/chemistry , DNA, Complementary/genetics , Entropy , Escherichia coli/chemistry , Escherichia coli/genetics , Genetic Vectors/chemistry , Genetic Vectors/genetics , Humans , Molecular Sequence Data , Molecular Weight , Oncogene Proteins/genetics , Protein Folding , Protein Structure, Secondary , RNA-Binding Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Homology, Nucleic Acid , Static Electricity , TemperatureABSTRACT
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.
