Human protein TGIF1 is an essential regulator of cell fate with broad roles in different tissues, and has been implicated in holoprosencephaly (HPE) and many cancers. The function of TGIFI in transcriptional regulation depends on its three-amino acid loop extension (TALE) type of homeodomain (HD). Two missense mutations that led to P192A and R219C substitutions in TGIFI-HD were previously found in HPE patients and suggested to be the causes for these cases. However, how these mutations affected TGIF1 function has not been investigated from a structural view. Here, we investigated the roles of P192 and R219 in TGIFI-HD structure packing through determining the NMR structure of TGIF1-HD. Surprisingly, P192 and R219 were found to play roles in packing alpha 1 and alpha 2 to alpha 3 together with A190 and F215 through side-chain interactions. Circular dichroism (CD) showed that P192A and R219C mutants displayed structural change and less folding compared with wild-type TGIFI-HD, and H-1-N-15 HSQC spectrum of P192A mutant exhibited chemical shift perturbations in all three helices of TGIFI-HD. Thus, it is suggested that P192A and R219C mutations led to structure disturbances of TGIF1-HD, which subsequently reduced the DNA-binding affinity of TGIF1-HD by 23-fold and 10-fold respectively, as revealed by the isothermal titration calorimetry (ITC) experiments. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and evidences for the roles of P192 and R219 in HD folding. (C) 2018 Elsevier Inc. All rights reserved.