The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

Abstract

Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys 45 and Cys 57 ). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys 45 and Cys 57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.Pax-8 is an important eukaryotic transcription factor that is responsible, during embryogenesis, for the differentiation of several organs such as kidney, thyroid, and neural tube. Pax-8 is also essential in the adult organism, where it activates thyroid hormone production. During development, Pax-8 functionality is regulated by alternative splicing, generating isoforms that exhibit different transactivation properties but maintain unaltered the DNA binding domain (1, 2). In mature thyroid follicular cells, the complete Pax-8 splicing isoform regulates the expression of thyroglobulin, thyroperoxidase (3, 4), sodium/ iodide symporter, and thyrotropin receptor genes (5-7). Pax-8 activity is co-regulated by thyroid transcription factor-1, thyroid transcription factor-2, and Hex, indicating an active role in the early commitment and differentiation of thyrocytes (8, 9). The Pax family shares a bipartite functionality consisting of a N-terminal binding region and a C-terminal transactivation region.The N-terminal region is usually comprised of three domains, namely a Paired Box (Prd) domain, a conserved octapeptide, and a further homeodomain (see Fig. 1). Differences in conservation and functionality in these domains have been used as evolution markers for the Pax family (10), which has been subdivided into four groups according to the sequence homology in the N-terminal region. In particular, the N-terminal region of Pax-8 is composed by all three domains but presents an incomplete and inactive homeodomain. The Prd domain consists of a well conserved 128-residue-long region, formed by two distinct subdomains known in literature as PAI (N-terminal) and RED (C-terminal) (11).DNA binding studies have demonstrated that the two subdomains of the Pax-8 Prd domain bind DNA independently (12), are both required f...

Pax factors in transcription and epigenetic remodelling

“…The two DNA interacting subdomains of PD are thus structurally related to the HD. Crystal structures of Drosophila Prd [13], PAX6 [14], and PAX5 [15] together with nuclear magnetic resonance analysis of PAX8 [16] have supported the model of the third helix of these HTH motifs being implicated in sequence-specific interactions. In addition, the N-terminal HTH motif, named PAI subdomain, is preceded by a ß-hairpin that acts as a clamp interacting with the phosphate backbone and minor groove.…”

Characterization of a novel loss‐of‐function mutation of PAX8 associated with congenital hypothyroidism

“…However, this amino acid represents a variant residue, being not conserved among the PAX genes 29 . It is worth noting that the structure of PAX8 bound to DNA has not been determined yet; therefore, any assessment on the PAX8–DNA complex has been made by comparison with other PAX homologues bound to DNA 31 . Our hypothesis is that the H55Q mutation within helix 3 of the paired domain is not involved in contacts with the DNA but might affect the folding and the conformation of the C‐terminal region of PAX8, possibly of domains of the protein known to be crucial for the transcriptional activity because involved in physical interactions with other coactivators and/or the basal transcriptional machinery.…”

The evolution of vision

“…The evolutionary origin of Pax6 is still largely unknown. So far Pax genes characterized by a paired box which encodes a bipartite DNA binding domain consisting of a PAI and a RED subdomain have only been found in multicellular metazoans and exclusively in bilateria. Both in mammals and insects there are approximately 9 or 10 Pax genes.…”