Abstract 18482: Structure Based Prediction of Stability and Affinity Changes in Human NKX2.5 HD Mutants

Abstract

Background: Cardiac transcription factor, NKX2.5, binds to the target DNA through its homeodomain (HD). To date, nearly 40 mutations in NKX2.5 have been found related to familial congenital heart disease. Homeodomain missense mutations have a high disease penetrance, with pleiotropic cardiac defects compared to missense mutations outside the homeodomain. In in vitro studies, the best correlation with clinical phenotypes and homeodomain mutation resulted from the markedly reduced DNA binding. We recently determined crystal structure of NKX2.5 HD bound to its natural DNA target. Using the structure, we sought to study whether DNA binding and folding energy changes in mutant proteins can be predicted through computational approaches. Methods and results: We used structure based energy prediction to calculate the binding energy and the stability of NKX2.5 mutant proteins. NKX2.5 HD from the high resolution crystal structure (PDB ID: 3RKQ ) was mutated to reflect the 14 genetic and 4 somatic point mutations identified in patients with cardiac anomalies. The HD-DNA models were energy minimized using the program Interface Model. This program utilizes a conservative approach by keeping the backbone structure of the protein and DNA intact while operating only on the sidechain rotamers to achieve the lowest energy state using monte-carlo simulation. Intra-protein and protein-DNA interaction energies of mutant models were calculated and the relative ΔG from the wild type was tabulated. Compared to the wild-type, all the genetic mutants showed reduced DNA binding while the somatic mutations show increased protein stability and DNA binding. The prediction is in good agreement with reported biochemical studies. Conclusion: The high resolution structure enables prediction of energy changes in NKX2.5 pathological mutants compared to wild type. The results well correlate with previously reported experimental data.