Structural and biochemical analysis of a B12 super-binder

The gut microbiota is pivotal to human health, playing an important role in nutrient absorption and immune system. A prominent bacterial family in the human gut is Bacteroidetes. Cobalamin (Vitamin B12) is an essential micronutrient for these gut bacteria. Here, we focused on the BtuG family proteins in B. thetaiotaomicron, a model organism for this family, particularly the three homologs BtuG1, BtuG2, and BtuG3, which serve as cobalamin scavengers. Our study aimed to understand the structural and biochemical attributes of these proteins to describe their function in cobalamin acquisition. We solved the crystal structures of all three BtuG homologs bound to different cobalamin forms and the precursor cobinamide, and measured the binding kinetics by the grained coupled interferometry (GCI) technology. Our results reveal high binding affinities, in the low picomolar range underlining their critical role in cobalamin scavenging. Leveraging the high-resolution crystal structures, we successfully designed mutants of these BtuG homologs, resulting in both increased and decreased binding affinities for cobalamin and its precursor. This study furthers our understanding of bacterial adaptation mechanisms in the gut environment, and reveals a family of proteins with unique characteristics toward B12 scavenging, with implications for medical and biotechnological research.