Structural analysis of runx1 protein and its mutants in complex with runx3 promoter shows altered conformations

Runt related transcription factor 1 also known as RUNX1 play a pivotal role in the regulation of the development of hematopoietic system in accordance with various transcriptional co-regulators. It is one of the most common targets of chromosomal translocations and mutations in its runt domain are frequently associated with leukemogenesis. Structural studies of RUNX1-DNA complexes provide details of direct contacts formed between runt domain of RUNX1 and DNA. The amino acid residues, Lys83 and Arg174, of RUNX1 directly interact with its binding site on the promoters. In present study, we have used a combined approach involving a detailed in-vitro and in-silico analysis of RUNX1 and its mutants for their structural and functional evaluation. CD spectroscopy and Tryptophan fluorescence of wild type and mutant full-length purified RUNX1 protein suggested an altered secondary and tertiary structure of mutant proteins. The mutant proteins also exhibited decrease in DNA binding as evident by Electrophoretic Mobility Shift Assays and binding kinetics using fluorescence spectroscopy. We observed that DNA binding affinity of mutated RUNX1 with RUNX3 promoter was about 5-7 fold lower than that of wild type RUNX1. These results suggest that both the point mutations (Lys83Glu/Arg174Gln) lead to a change in conformation of full-length RUNX1 protein which in turn affects its binding to DNA. Investigations of the molecular insights using in-silico approach suggest that this decrease in DNA binding could be due to changes in hydrogen bonding pattern, and lengths between wild type and mutant protein complexed with RUNX3 promoter.