Published online 10 May 2005
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The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions
Biochemistry Department, Institute of Medical Biology, University of Tromsø 9037 Tromsø, Norway 1Department of Pharmacology, Institute of Medical Biology, University of Tromsø 9037 Tromsø, Norway 2Department of Pharmacology, Institute of Pharmacy, University of Tromsø 9037 Tromsø, Norway
*To whom correspondence should be addressed. Tel: +47 776 44720; Fax: +47 776 45350; Email: terjej{at}fagmed.uit.no
Received February 10, 2005. Revised April 20, 2005. Accepted April 20, 2005.
The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6
PD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein–protein interactions, but not DNA binding of Pax6PD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein–protein interactions.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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