Solution structure determination of core binding factor beta(1--141) and map of CBF-alpha binding site.

摘要

Core binding factor (CBF) is a heterodimeric transcription factor that is essential for a number of developmental process including hemotopoiesis and bone development. CBFs contain a DNA-binding CBF a subunit and a non-DNA binding CBF b subunit that increases the binding affinity of CBF a for DNA. Homozygous disruption of either the Cbfa2 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the CBFA2 and CBFB genes are associated with a variety of acute leukemias. The CBFA1 gene, which encodes another CBF a subunit, is required for bone development, and mutations in one copy of the CBFA1 gene are associated with an autosomal dominant skeletal disorder, Cleidocranial dysplasia. The lack of homology of either of the subunits of CBF to other known proteins makes them important targets for structure determination.; We have developed a procedure for overexpressing and purifying full-length CBF b as well as a truncated form of CBF b (1-141) protein using a novel glutaredoxin fusion expression system. The N-terminal 141 amino acids of CBF b contains the heterodimerization domain for the DNA-binding CBF a subunits and binds to CBF a in vitro with the same affinity as the full length CBF b . The high resolution three-dimensional structure of CBF b (1-141) was determined by NMR. It is a novel a/b structure consisting of two 3-stranded b -sheets packed on one another in a sandwich arrangement, with four peripheral a -helices. The CBF a binding site on CBF b was mapped by chemical shift perturbation analysis. The interaction between CBF a and CBF b thus provides another example of the importance of peptide surface association in proteins involved in transcription regulation. The fold of the central six-stranded b -sheet has some similarities to that observed in SH3 domains.