title: A mutation in the S-switch region of the Runt domain alters the dynamics of an allosteric network responsible for CBFbeta regulation.
abstract: The Runt domain is the DNA binding domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFbeta subunit heterodimerizes with the Runt domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFbeta allosterically regulates DNA binding by quenching conformational exchange in the Runt domain, particularly in the S-switch region and the betaE'-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the betaE'-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt domain mutant, R164N, binds both DNA and CBFbeta with higher affinity, but it is less sensitive to allosteric regulation by CBFbeta. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFbeta and DNA binding sites on the Runt domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lukasik
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Liu
authors.forename: Jolanta
authors.initials: J
authors.lastname: Grembecka
authors.forename: Izabela
authors.initials: I
authors.lastname: Bielnicka
authors.forename: John H
authors.initials: JH
authors.lastname: Bushweller
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Speck
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D050657:Core Binding Factor alpha Subunits / Q000235:genetics / Q000378:metabolism*; D050658:Core Binding Factor beta Subunit / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation
chemical_list: C493692:CBFB protein, human; D050657:Core Binding Factor alpha Subunits; D050658:Core Binding Factor beta Subunit; D004247:DNA