Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

Abstract

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

Protein metalation in biology

“…This article presents an equilibrium thermodynamic framework for biological metalation which in turn will provide the context for understanding the magnitude of kinetic contributions. Notably, this approach alone largely explained metal specificity in the handful of systems examined to date and, in some cases, identified disparities that informed further experimentation into metal specificity, for example, into the contributions of adducts with nucleotide cofactors [ 7∗ , 8∗∗ , 9∗∗ ].…”

“…Differences in free energy for metal complex formation with Mg 2+ GTP-CobW versus the intracellular milieu (ΔΔ G ) in an ideal cell where sensors are at their midpoints identify favourable (red, also on panel b) or unfavourable (blue) thermodynamic gradients for metalation. A metalation calculator compares such values to calculate in vivo metal occupancies [ 9 ]. …”

“…By comparing the metal affinities of proteins, also expressed as free energies for complex formation, with these values, in vivo metalation becomes fathomable [ 8 ]. For example, a GTPase involved in Co 2+ supply for vitamin B 12 biosynthesis, cobalamin operon gene W (CobW), is shown to outcompete the cytosol for Co 2+ and for Zn 2+ when bound to Mg 2+ GTP (and when the sensors are at their midpoints of their dynamic ranges in idealised cells) [ 9 ]. Figure 3 b thus shows that the thermodynamic gradient for ligand exchange to Mg 2+ GTPCobW is favourable (negative shown in red) for Co 2+ and for Zn 2+ but unfavourable (positive shown in blue) for other metals even though the protein binds Cu + most tightly.…”

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

“…Two Escherichia coli strains, ED656 and ED662 (ΔbtuF), were also used as B 12 -producers. E. coli strain ED656 was constructed in a similar fashion to ED741 (Young et al ., 2021). ED656 was generated from E. coli MG1655 by engineering it to contain a set of B 12 biosynthesis genes.…”

SARS-CoV-2 Mproinhibition by a zinc ion: structural features and hints for drug design

“…[17][18][19][20] Furthermore, zinc ions are not homogeneously distributed within the cells, with large differences among cellular compartments and organelles. 21,22 Zinc has a key role in the signalling pathways of the innate and adaptive immune reactions. 23,24 It has been reported that zinc is able to participate in many processes of defense against infections.…”