Modo de acción molecular de CarH, el prototipo de una nueva familia de fotorreceptores dependientes de la vitamina B12plasticidad en su oligomerización, unión al DNA y diseño de su operador

Laburpena

Living organisms employ photoreceptor proteins to respond to light. A recently discovered family of bacterial photoreceptors sense light using as chromophore the vitamin B12 form known as 5?- deoxyadenosylcobalamin (AdoCbl), a finding that unveiled a novel biological facet of this vitamin, that as a light-sensor. The prototype of this family, CarH, is a transcriptional repressor that usually regulates light-dependent expression of its own gene, and of those involved in the synthesis of carotenoids that help mitigate photooxidative damage. The molecular mechanism of action is best understood for the Thermus thermophilus homolog, TtCarH. ApoTtCarH is a monomer that, in the dark, binds to AdoCbl to form a tetramer, which binds to operator DNA and represses transcription. Light disrupts AdoCbl and thereby the tetramer to monomers unable to bind operator DNA. Structures of the TtCarH tetramer, free and bound to DNA, and of the light-exposed monomer provided atomic level insights into its architecture and mode of action. This was corroborated by extensive mutational and in vitro analysis. TtCarH is composed of an autonomous N-terminal winged-helix DNA binding domain (DBD) similar to those in MerR proteins. It is connected by a flexible linker to an autonomous AdoCbl-binding, light-sensing, oligomerization domain comprising two subdomains: a four-helix bundle and a Rossmann-fold. Although very similar in structure to the methionine synthetase methylcobalamin-binding module, the CarH AdoCbl-binding domain binds to the larger AdoCbl, and this depends crucially on a W-x(9)-EH motif that is highly conserved among CarH homologs. Two AdoCbl-bound C-terminal domains pack as head-to-tail dimers, and two such dimers assemble as tetramer in the dark in which the four DBDs are splayed around the surface. This results in a unique mode of DNA binding in which the TtCarH tetramer uses three of its four DBDs to contact three contiguous 11 base-pair direct repeats (DRs) with a consensus 5'-nAnnTnnACAn-3' sequence. The operator overlaps with the -35 element of a promoter recognized by RNA polymerase holoenzyme containing the major A factor. Photolysis of TtCarH-bound AdoCbl releases the 5'-deoxyadenosyl group and produces a large shift in the four-helix bundle relative to the Rossmann fold to provoke tetramer disassembly and loss of DNA binding. Work in this thesis also describes a second homolog, BmCarH, from the Gram-positive bacterium Bacillus megaterium. ApoBmCarH is an oligomeric molten globule that does not bind DNA. However, AdoCbl binding in the dark results in a BmCarH tetramer with the proper fold for DNA-binding, and the conserved W-x(9)-EH motif is crucial for this. The tetramer is again disrupted by light but to dimers and not monomers. Analysis of BmCarH-DNA binding revealed an operator design similar to that for TtCarH, which overlaps with the -35 or the -10 promoter element and is larger by an additional 11 base pair DR. Interestingly, BmCarH could bind to the three-repeat TtCarH operator, and TtCarH to three contiguous DRs of the four-repeat CarH operator. This DNA binding adaptability of BmCarH may stem from its longer linker between the DNA and AdoCbl-binding domains. Altogether, there is a remarkable plasticity in the oligomerization, operator architecture and DNA binding among CarH photoreceptors within an overall conserved mode of action, which is relevant for its biological function and use as an optogenetics tool.