Structural and Biophysical Insights into the Ligand-Free Pitx2 Homeodomain and a Ring Dermoid of the Cornea Inducing Homeodomain Mutant

Abstract

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. NMR techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes the autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA-binding affinity and a 5°C decrease in the thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in only a 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to other Pitx2 homeodomain mutations.

Identification of a novel frameshift mutation in PITX2 gene in a Chinese family with Axenfeld-Rieger syndrome

“…PITX2 is a bicoid-related homeodomain transcription factor required for the development and maintenance of the ocular anterior segment tissues and nonocular tissues including the pituitary, heart, and branchial arches (Gage et al, 1999;Kitamura et al, 1999;Kelberman et al, 2011;Doerdelmann et al, 2012). PITX2 is dosage-sensitive and either increased or decreased alteration causes developmental disorders, such as ARS and glaucoma (Tümer and BachHolm, 2009).…”

“…The 60-amino acid homeodomain consists of three helices in a compactly folded structure and a flexible N-terminal arm, responsible for the recognition and binding to target DNA sequences (Doerdelmann et al, 2012). Helix 3, also called the recognition helix, inserts itself into the major groove of the recognition site and establishes contacts with the specific residues of the bicoid binding sequence (Li et al, 2008;Doerdelmann et al, 2012). Helix 2 makes DNA backbone contacts, enhancing specific homeodomain-DNA interactions.…”

“…Helix 2 makes DNA backbone contacts, enhancing specific homeodomain-DNA interactions. In addition to the helices, the N-terminal arm makes contacts with the bases in the DNA minor groove (Doerdelmann et al, 2012).…”

“…4 and 5). Helix 2 and helix 3, the crucial helical regions of the homeodomain, are essential for binding DNA and are critical for the transcription factor function (Dave et al, 2000;Doerdelmann et al, 2012). In addition, the OAR domain in the C-terminal end has been proposed to increase the DNA binding and transcriptional activity of PITX2 during development (Amendt et al, 2000).…”

“…The structure was determined using nuclear magnetic resonance (NMR) spectroscopy (Doerdelmann et al, 2012).…”