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

Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and func tion. Site-directed mutagenesis; allows specific hypotheses to be test ed, but its systematic application to entire structural motifs is inef ficient. This limitation may be overcome by genetic selection, which a llows rapid scoring of thousands of randomly (or pseudorandomly) gener ated mutants. To facilitate structural dissection of DNA-binding prote ins, we have designed two generally applicable bacterial selection sys tems: pPLUS selects for the ability of a protein to bind to a user-def ined DNA sequence, whereas pMINUS selects against such binding. Comple mentary positive and negative selections allow rapid mapping of critic al residues. To test and calibrate the systems, we have investigated t he bipartite POU domain of the human B-cell-specific transcription fac tor Oct-2. (i) An invariant residue (Asn347) in the DNA-recognition he lix 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 selectio n and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems. An essentia l role for Asn347 in wild-type POUHD-DNA recognition is consistent wit h homologous Asn-adenine interactions in cocrystal structures of canon ical 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 w ith 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.