Abstract
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.
Highlights
Vitamin B12 is the largest and most structurally complex vitamin in nature.[1]
The position of AdoCbl between the Rossman fold and the 4-helix bundle places the upper axial 50-deoxyadenosyl ligand (Ado) in steric contact with W131 from the 4-helix bundle (Fig. S1b†).[10]. This is thought to force each holo-TtCarH monomer to adopt an upright conformation that facilitates the formation of head-to-tail dimers
In both Grampositive and Gram-negative bacteria, AdoCbl negatively regulates its own biosynthesis independently of light by directly binding to regions of messenger RNA known as riboswitches.[23]
Summary
Vitamin B12 is the largest and most structurally complex vitamin in nature.[1]. At its center is a highly conjugated cobalamin macrocycle, which enables one active derivative, 50-deoxyadenosylcobalamin (AdoCbl, Fig. S1a†), to act as a latent source of radicals for numerous mutase[2] and eliminase[3] enzymes.
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