Structure of a new DNA-binding domain which regulates pathogenesis in a wide variety of fungi

Abstract

WOPR-domain proteins are found throughout the fungal kingdom where they function as master regulators of cell morphology and pathogenesis. Genetic and biochemical experiments previously demonstrated that these proteins bind to specific DNA sequences and thereby regulate transcription. However, their primary sequence showed no relationship to any known DNA-binding domain, and the basis for their ability to recognize DNA sequences remained unknown. Here, we describe the 2.6-Å crystal structure of a WOPR domain in complex with its preferred DNA sequence. The structure reveals that two highly conserved regions, separated by an unconserved linker, form an interdigitated β-sheet that is tilted into the major groove of DNA. Although the main interaction surface is in the major groove, the highest-affinity interactions occur in the minor groove, primarily through a deeply penetrating arginine residue. The structure reveals a new, unanticipated mechanism by which proteins can recognize specific sequences of DNA.fungal pathogenesis | transcription factor | transcriptional regulation | protein-DNA interaction | Candida albicans T he WOPR family of transcriptional regulators [named for the members Wor1 (white-opaque regulator 1), Pac2 (pat1 compensator 2), and Ryp1 (required for yeast phase growth 1)] controls morphological changes and pathogenesis in a diverse group of fungal species (Fig. 1A), including the human pathogens Candida albicans (1-3), Histoplasma capsulatum (4), and Cryptococcus neoformans (5) and several plant pathogens (6-9). In C. albicans, Wor1 controls the process of white-opaque switching and mating (1-3), and it has a key role in promoting commensalism (10). In H. capsulatum, it controls the transition from the mycelia form (found in soil) and the yeast form (found in infected human hosts) (4). Recently, a series of in vitro and in vivo experiments demonstrated that the WOPR domain of Wor1 binds DNA in a sequence-specific fashion and defined the DNA sequence recognized by it (Fig. 1B) (11,12). Muc1 expressed independent of TEC1 1 (Mit1) from Saccharomyces cerevisiae, a fourth WOPR-domain protein, and Ryp1 from H. capsulatum have since been shown to recognize this same DNA sequence, representing conservation of the WOPR domain-DNA sequence interactions over a period of 600 million to 1.2 billion years (13).Biochemical experiments indicated that the WOPR domain binds DNA in an unusual way. The WOPR domain consists of two regions conserved across many fungal species (referred to here as "R1" and "R2"), separated by a poorly conserved "linker" region of variable length (Fig. 1C). R1 is 80 aa in length, R2 is 50 aa, and the linker ranges from less than 25 to more than 100 aa, depending on the species. The WOPR domain alone can bind DNA tightly (∼5 nM) and specifically (11). Additional experiments demonstrated that neither R1 nor R2 alone could bind to DNA individually, but binding was observed-albeit at lower affinity-when the two regions were expressed as separate peptides and mixed in the presence of DNA. ...

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

“…The DNA binding site and protein crystal structure of several homologs of Sge1 have been resolved [33,41–43]. The amino acids interacting with DNA are highly conserved among Wor1/Sge1 homologs from different fungi [23,27,29,30,41,43,44]. Consistent with this, the DNA binding sites of Sge1 orthologs in Saccharomyces cerevisiae (Mit1) and the filamentous fungus Histoplasma capsulatum (Ryp1) are the same as for Wor1 [33].…”

Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae

“…MOT3 + ), while in the case of the mit1 alleles, the difference is clearer as mit1D0 confers the same phenotype as MIT1 + . Both the mot3-1 and mit1-1 mutations encode changes in the DNA binding domains of these transcription factors (Madison et al 1998;Cain et al 2012), as mot3-1 causes an N388H change in a position that directly contacts DNA (Grishin et al 1998), while mit1-1 causes an H187R change at a position that likely causes a conformational change of the DNA binding domain (Lohse et al 2014) (M. B. Lohse and A. D. Johnson, personal communication).…”

The WOR1 5′ untranslated region regulates white‐opaque switching in Candida albicans by reducing translational efficiency

“…The master regulator of white‐opaque switching is WOR1 , which is highly expressed in opaque cells and required for switching to opaque (Huang et al ., ; Srikantha et al ., ; Zordan et al ., ). Wor1 protein contains a novel DNA‐binding domain (Lohse et al ., 2010; 2014; Zhang et al ., ) and promotes its own expression by binding to the WOR1 promoter up to 8 kb upstream of its transcription start site (Zordan et al ., ). White‐opaque switching and WOR1 transcription are regulated by a circuit of interlocking transcriptional feedback loops, consisting of regulators WOR1 , EFG1 , CZF1 , WOR2 , WOR3 and AHR1 (Downs et al ., ; Levchenko and Jackson, ; Zordan et al ., ; Hernday et al ., ; Lohse et al ., ).…”