MAPPING CRITICAL RESIDUES IN EUKARYOTIC DNA-BINDING PROTEINS - A PLASMID-BASED GENETIC SELECTION STRATEGY WITH APPLICATION TO THE OCT-2 POU MOTIF

Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and function. Site-directed mutagenesis; allows specific hypotheses to be tested, but its systematic application to entire structural motifs is inefficient. This limitation may be overcome by genetic selection, which allows rapid scoring of thousands of randomly (or pseudorandomly) generated mutants. To facilitate structural dissection of DNA-binding proteins, we have designed two generally applicable bacterial selection systems: pPLUS selects for the ability of a protein to bind to a user-defined DNA sequence, whereas pMINUS selects against such binding. Complementary positive and negative selections allow rapid mapping of critical residues. To test and calibrate the systems, we have investigated the bipartite POU domain of the human B-cell-specific transcription factor Oct-2. (i) An invariant residue (Asn347) in the DNA-recognition helix bf the POU-specific homeodomain (POUHD) was substituted by each of the 19 other possible amino acids. The mutant proteins each exhibited decreased specific DNA binding as defined in vivo by genetic selection and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems. An essential role for Asn347 in wild-type POUHD-DNA recognition is consistent with homologous Asn-adenine interactions in cocrystal structures of canonical homeodomains. (ii) Extension of pPLUS/pMINUS selection to the POU-specific subdomain (POUS) is demonstrated by analysis of mutations in its putative helix-turn-helix (HTH) element. The altered DNA-binding properties of the mutant proteins in vivo and in vitro are in accord with a structural analogy between POUS and the operator-binding domain of phage lambda repressor. Together, our results provide a foundation for future biochemical studies of the structure and evolution of POUHD and POUS HTH sequences.