BIOCHEMICAL-CHARACTERIZATION OF THE OCT-2 POU DOMAIN WITH IMPLICATIONS FOR BIPARTITE DNA RECOGNITION

B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins. A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2. Octamer recognition is mediated by the POU domain, a conserved structural motif which-like the zinc finger and leucine zipper-defines a family of related transcription factors. Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence. As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin. Biochemical and biophysical characterization yields the following results. (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity. (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker. (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers. (iv) Unlike the intact POU domain, the isolated subdomains do not exhibit high-affinity binding to the octamer DNA site, nor is DNA binding restored by mixing the two isolated subdomains. (v) Circular dichroism studies demonstrate that the intact POU-specific region and homeodomain each contain substantial alpha-helix and exhibit cooperative folding/unfolding transitions. The alpha-helix content of the intact POU region is equal to the sum of the alpha-helix content of the two subdomains. (vi) H-1-NMR spectra of the isolated subdomains are additive to yield a spectra that is nearly identical to that of the parent POU domain. Together, these results confirm the bipartite structure of the POU domain and the existence of the POU-specific and variant homeo-subdomains as autonomous structural units.