Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.     

Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain.

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBF alpha, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the beta subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-kappaB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD-beta-DNA ternary complex identified the surface of the RD in contact with the beta subunit, suggesting a mechanism for the enhancement of RD DNA binding by beta. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.     

Altered affinity of CBF beta-SMMHC for Runx1 explains its role in leukemogenesis

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBF beta), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBF beta fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBF beta-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBF alpha) with higher affinity than the native CBF beta protein. NMR studies identify interactions in the CBF beta portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.     

Structure/function analysis of the integrin beta 1 subunit by epitope mapping

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti- chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.