A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

Abstract

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor

“…2). Overall, the structural features of the Rep-Ant complex described here are different from the structural features of other known Rep-Ant complexes (2,16).…”

“…To initiate the expression of desired genes, Rep-operator interactions must be disrupted, which can occur by various means, including the binding of small molecule inducers (2,3). Interactions with these components disrupt the ability of a Rep to bind to the operator by either altering its 3D structure or inducing proteolysis (4).…”

Light-dependent gene regulation by a coenzyme B 12 -based photoreceptor

“…Some of these homologues may, like CarH, depend on B 12 for function, whereas others, like CarA, may not. It is worth noting that, other than in Stigmatella aurantiaca (a myxobacterium very closely related to M. xanthus), CarS homologues are not found (12,18). Although factors resembling CarS in function, but not in sequence, may exist for on/off control of some of the newly identified CarA/CarH homologues, a more straightforward possibility is that the activities of many of these homologues are modulated solely by a lightresponsive AdoB 12 switch, like the one identified in this study.…”

“…We next examined the functional relevance of AdoB 12 photosensitivity on P B expression. For this, we used a ΔcarS M. xanthus genetic background, to separate the direct effect of light on AdoB 12 -CarH from antirepression by CarS (12,18). Consistent with CarA being B 12 -independent and with the need for CarS to counteract its activity, the strain in which P B is repressed only by CarA (ΔcarS ΔcarH) remained yellow in the light with or without B 12 (Fig.…”

“…S1A) (13). CarS is an antirepressor with an SH3-domain topology that mimics operator DNA to specifically target the DNA recognition helix of the MerR-type DNA-binding winged-helix domain of two paralogous repressors, CarA and CarH, to counteract their binding to operator (18,19). In the dark, these two repressors down-regulate the promoter P B that drives expression of the carB-carA gene cluster, where the remaining carotenogenic genes are grouped (Fig.…”