The Hox proteins increase DNA-binding specificity through dimerization with TALE-type, nonMetaHox homeodomain proteins, like Pbx. As the ancestral Msx protein includes a TALE-interaction domain, we sought to test its interaction with MSX1 enhancer sequences utilized within vertebrate facial prominences, containing putative canonical binding sites.
Objective: To validate and characterize the binding interactions between the MSX1, PBX1 proteins and MSX1 enhancer double stranded DNA (dsDNA) using various biophysical techniques.
Methods: Wild-type and mutant versions of MSX1 and PBX1 were cloned into the Gateway system (Invitrogen) in multiple truncated versions and expressed as soluble protein in Esherichia coli. The proteins were purified to homogeneity and appropriate nucleotide sequences were annealed and incubated together, before being subjected to protein crystallization and isothermal titration calorimetry (ITC) experiments.
Results: We have obtained pure MSX1 protein (amino acids 132-242) in large (milligram) quantities and have crystals of MSX1 bound to dsDNA from its own upstream distal enhancer region. We have determined binding affinities using ITC between MSX1 and dsDNA-1 (5’-TGATTGGTCTAATTGC-3’) in the low micromolar range.
Conclusions: MSX1 interacts strongly with its own upstream distal enhancer region. Biophysical characterization of this system will help outline the interactions between MSX1, PBX1, and relevant DNA sequences, as well as determine the molecular mechanisms underpinning MSX1 transcriptional activity.
Keywords: Biophysics, Cleft lip-palate, Oral biology, Proteins and structural biology
See more of: Craniofacial Biology