Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility.

Abstract

Pit-1, a member of the POU domain family of transcription factors, characterized by a bipartite DNA-binding domain, serves critical developmental functions based on binding to diverse DNA elements in its target genes. Here we report a high resolution X-ray analysis of the Pit-1 POU domain bound to a DNA element as a homodimer. This analysis reveals that Pit-1 subdomains bind to perpendicular faces of the DNA, rather than opposite faces of the DNA as in Oct-1. This is accomplished by different spacing and orientation of the POU-specific domain. Contrary to previous predictions, the dimerization interface involves the carboxyl terminus of the DNA recognition helix of the homeodomain, which in an extended conformation interacts with specific residues at the amino terminus of helix al and in the loop between helices a3 and a4 of the POU-specific domain of the symmetry related monomer. These features suggest the molecular basis of disease-causing mutations in Pit-1 and provide potential basis for the flexible allostery between protein domains and DNA sites in the activation of target genes.[Key Words: Crystal structure; Pit-1; POU-domain factors; dimerization; spacing; orientation] Received October 25, 1996; revised version accepted December 3, 1996.The expression of specific genes in cells is often governed by families of transcription factors harboring DNA-binding motifs. In eukaryotes, these factors often exhibit extraordinary versatility. For instance, the bZip and helix-loop-helix families of transcription factors bind to DNA as both homo-and heterodimers (Bexevanis and Vinson 1993;Glover and Harrison 1995). The nuclear receptors go a step further and recognize DNA elements with different spacings and polarities between the half-sites (Naar et al. 1991;Umesono et al. 1991;Kurokawa et al. 1993). The POU domain family of transcription factors are perhaps the most remarkable in their ability to bind DNA as both monomers and dimers and in their ability to adopt diverse configurations (Wegner et al. 1993;Herr and Cleary 1995). Part of this versatility derives from their unusual bipartite structure, consisting of a highly conserved -75 amino acid POUspecific domain (POUg), tethered by a linker of variable length and composition, to a 60-amino-acid homeodomain (POUH). The flexibility of the linker, in particular, allows the possibility of different relative spacings and orientations between the subdomains. This helps to exThese authors contributed equally to this work. "Corresponding author. Present address:

Malleability of folding intermediates in the homeodomain superfamily

“…The known crystal structure of Pit1 is from a complex with DNA (13). We checked the solution structure of the free protein by residual I8V S9A A12G K13A D14A D14G A15G R18A R18G F20A E22G S27A S28G{ I31V M32A M32G A35G E37A E37G N39A L40V E41A E41G K42A E43A E43G V44A F49A S50A I8V S9A A12G K13A D14A D14G A15G R18A R18G F20A E22G S27A S28G{ I31V M32A M32G A35G E37A E37G N39A L40V E41A E41G K42A E43A E43G V44A F49A S50A I8V S9A A12G K13A D14A D14G A15G R18A R18G F20A E22G S27A S28G{ I31V M32A M32G A35G E37A E37G N39A L40V E41A E41G K42A E43A E43G V44A F49A S50A S50G PhiTS2 2M PhiTS2 Global Phi TS2 Various mutations were introduced to probe different interactions (Table S3).…”

PBX proteins: much more than Hox cofactors

“…Although this aspect of Pbx homodimerization has not been further explored, it could play a role in gene regulation through a competition between various partners. Indeed, several studies have addressed the importance of homodimerization for other homeodomain-containing transcription factors such as Oct1 (Poellinger and Roeder, 1989), Paired (Wilson et al, 1993), Cdx2 (Suh et al, 1994), Evenskipped (Hirsch and Aggarwal, 1995), Mix1 (Mead et al, 1996) and Pit1 (Jacobson et al, 1997). Homodimerization of Pbx proteins might be a way of preventing their interaction with other transcription factors and their binding onto certain gene promoters.…”

Characterization of Homo- and Heterodimerization of Cardiac Csx/Nkx2.5 Homeoprotein

“…However, a very limited number of studies have addressed the homo-and heterodimerization of HD-containing transcription factors among more than 400 members of HD proteins from yeast to human. Homodimerization ability has been demonstrated for Oct1 (10), Paired (11), Cdx2 (12), Even-skipped (13), Mix.1 (14), and Pit1 (15). Heterodimerization was shown for HNF1␣-HNF1␤ (16), Oct1-Oct2 (15), Mix.1-Siamois (14), MCM1-MAT␣2 (17,18), and Extradenticle-Ultrabithorax (19).…”