Distinct class of DNA-binding domains is exemplified by a master regulator of phenotypic switching in Candida albicans

Abstract

Among the most important classes of regulatory proteins are the sequence-specific DNA-binding proteins that control transcription through the occupancy of discrete DNA sequences within genomes. Currently, this class of proteins encompasses at least 37 distinct structural superfamilies and more than 100 distinct structural motifs. In this paper, we examine the transcriptional regulator Wor1, a master regulator of white-opaque switching in the human fungal pathogen Candida albicans. As assessed by a variety of algorithms, this protein has no sequence or structural similarity to any known DNA-binding protein. It is, however, conserved across the vast fungal lineage, with a 300aa region of sequence conservation. Here, we show that this 300aa region of Wor1 exhibits sequence-specific DNA binding and therefore represents a new superfamily of DNA-binding proteins. We identify the 14-nucleotide-pair DNA sequence recognized by Wor1, characterize the site through mutational analysis, and demonstrate that this sequence is sufficient for the Wor1-dependent activation of transcription in vivo. Within the 300aa DNA-binding conserved region, which we have termed the WOPR box, are two domains (WOPRa and WOPRb), dissimilar to each other but especially well-conserved across the fungal lineage. We show that the WOPR box binds DNA as a monomer and that neither domain, when expressed and purified separately, exhibits sequence-specific binding. DNA binding is restored, however, when the two isolated domains are added together. These results indicate that the WOPR family of DNA-binding proteins involves an unusual coupling between two dissimilar, covalently linked domains.DNA-protein interaction | transcription factor | gene expression | DNA motif | heritable states of transcription

Crystal structure of the WOPR-DNA complex and implications for Wor1 function in white-opaque switching of Candida albicans

“…These results can be well explained by the structural data as these nucleotides are recognized by WOPR via hydrophilic and hydrophobic interactions and most of the substitutions would either disrupt favorable interactions or cause unfavorable steric hindrances with the protein. The structural and biochemical data together indicate that the WOPR domain can recognize and bind a dsDNA containing the characteristic motif TAAACT with a strictly conserved T at position 1 and a highly conserved T at position 6 but some variations at the other positions with base preferences, which are largely in agreement with the biochemical results by Lohse et al [23]. This finding may explain why there are so many different CaWor1-binding sites (nearly 200 sites) in the C. albicans genome [15].…”

“…Previous biochemical data showed that the N-terminal region of CaWor1 exhibits sequence-specific DNA-binding ability and can recognize and bind specifically to six different DNA sequences corresponding to the promoter regions of three genes [23]. To carry out the structural studies of CaWor1 in complex with its specific DNA, we first constructed the entire WOPR box of CaWor1 (residues 1-325) into the pET28a expression plasmid with the two CUG codons (residues Ser60 and Ser200) converted to UCG to compensate for the alternative genetic code in C. albicans [25] and expressed it in E. coli.…”

“…It is predicted to consist of two globular domains (WOPRa and WOPRb), dissimilar to each other, but well-conserved across the fungal lineage, connected by a low-complexity insertion [23,24]. The WOPR box binds DNA as a monomer and Wor1 binding requires the presence of both WOPRa and WOPRb [23]. However, how WOPRa and WOPRb interact with each other and bind to the specific DNA in a concerted way is unknown.…”

“…The conserved WOPR box is required for binding specific DNA sequences from different promoter regions and the binding is sufficient for the Wor1-dependent transcriptional activation [23]. It is predicted to consist of two globular domains (WOPRa and WOPRb), dissimilar to each other, but well-conserved across the fungal lineage, connected by a low-complexity insertion [23,24]. The WOPR box binds DNA as a monomer and Wor1 binding requires the presence of both WOPRa and WOPRb [23].…”

“…CaWor1 is a transcription regulator of 785 residues, consisting of a conserved N-terminal region (residues 1-325) termed WOPR box and a non-conserved and functionally and structurally undefined C-terminal region [23]. The conserved WOPR box is required for binding specific DNA sequences from different promoter regions and the binding is sufficient for the Wor1-dependent transcriptional activation [23]. It is predicted to consist of two globular domains (WOPRa and WOPRb), dissimilar to each other, but well-conserved across the fungal lineage, connected by a low-complexity insertion [23,24].…”