Pax proteins are transcriptional regulators that play important roles durin
g embryogenesis, These proteins recognize specific DNA sequences via a cons
erved element: the paired domain (Prd domain). The low level of organized s
econdary structure, in the free state, is a general feature of Prd domains;
however, these proteins undergo a dramatic gain in alpha-helical content u
pon interaction with DNA ('induced fit'). Pax8 is expressed in the developi
ng thyroid, kidney and several areas of the central nervous system. In huma
ns, mutations of the Pax8 gene, which are mapped to the coding region of th
e Prd domain, give rise to congenital hypothyroidism. Here, we have investi
gated the molecular defects caused by a mutation in which leucine at positi
on 62 is substituted for an arginine. Leu(62) is conserved among Prd domain
s, and contributes towards the packing together of helices 1 and 3. The bin
ding affinity of the Leu(62)Arg mutant for a specific DNA sequence (the C s
equence of thyroglobulin promoter) is decreased 60-fold with respect to the
wildtype Pax8 Prd domain. However, the affinities with which the wild-type
and the mutant proteins bind to a non-specific DNA sequence are very simil
ar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8
and the Leu(62)Arg mutant possess a low alpha-helical content; however, in
the Leu(62)Arg mutant, the gain in alpha-helical content upon interaction
with DNA is greatly reduced with respect to the wild-type protein. Thus the
molecular defect of the Leu62Arg mutant causes a reduced capability for in
duced fit upon DNA interaction.