The solution structure of DNA-free Pax-8 paired box domain accounts for redox regulation of transcriptional activity in the pax protein family.
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 redox-based mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys45 and Cys57). 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 Cys45 and Cys57 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.
Codutti
Luca
L
Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Udine, p.le Kolbe 4, 33100 Udine, Italy.
van Ingen
Hugo
H
Vascotto
Carlo
C
Fogolari
Federico
F
Corazza
Alessandra
A
Tell
Gianluca
G
Quadrifoglio
Franco
F
Viglino
Paolo
P
Boelens
Rolf
R
Esposito
Gennaro
G
D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D051757:PAX5 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000502:physiology
D005136:Eye Proteins; D018398:Homeodomain Proteins; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D004247:DNA