Interplay between chromophore binding and domain assembly by the B₁₂-dependent photoreceptor protein, CarH

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B₁₂-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B₁₂ to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B₁₂ photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B₁₂-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B₁₂. When B₁₂ is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B₁₂ is scarcer, as is likely in nature, tetramers with native-like structures can form without a B₁₂ complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B₁₂ shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B₁₂-dependent photoreceptors as light-activated tools for biomedical applications.