Introduction to ModG

ModG is a cytoplasmic molybdate-binding protein exclusive to the aerobic nitrogen-fixer Azobacter vinelandii.^[1] Molybdate is a molybdenum oxyanion (MoO42-). The group 6 element molybdenum is required by many enzymes that catalyze reactions associated with carbon, nitrogen, or sulfur metabolism.^[2] It is also part of the cofactor of the molybdoenzyme ModG. Not surprisingly, studies have linked ModG to molybdenum homeostasis within the cell.^[1]

Structure

The molybdoenzyme is a homotrimer.^[1] It can bind up to 8 molybdate molecules between 4 different types of on subunit interfaces (BS1, BS1’, BS2, and BS2’). Binding site 1 and binding site 2 are found at opposite ends of the protein; binding site 1’ and binding site 2’ are found off-axis near like ends.^[3] The sites are connected by hydrogen bonds and thus a cooperative binding mechanism has been proposed for ModG whereby ligand engagement with type 2 sites induces conformational changes to asparagine residues at type 1 sites, reading the site for ligand interactions.^[4] The structure of ModG was solved by Delarbe et al. using multi-wavelength anomalous dispersion (MAD).^[5] Crystallization required salt-free conditions established with polyethylene glycol (PEG), at which point the authors solved the PEG crystal form using molecular replacement.^[5]

The protein is made up of 3 identical subunits that have 67 amino acid pairs and are 14.3 kDa in size.^[1] In trimer form the subunits are perpendicular to each other and intersect at the 3-fold axis (as seen in the crystallized form).^[3] Each subunit is composed of two β-barrel domains (Domain I and Domain II) that each feature a short 310-helix.^[3] Each β-barrel domain includes five antiparallel β-strands arranged in a motif that is capped by two-turn α-helices.^[3] The folding arrangement of the ModG domains is that of an oligomer-binding (OB) fold, characteristic of toxins and other intracellular oxyanion-binding proteins.^[3] In trimerization, N and C termini (both found in Domain I) of a subunit interact with the β5-β6 loop (Domain II) of the adjoining subunit by sharing antiparallel β-sheets. Approximately 40% of monomer surface area is buried when the protein is in trimer form.^[1]

Properties and binding affinity

About 60% of each subunit interface is (40% is polar); furthermore, there is an unequal charge distribution that is attributed to the presence of 4 side chains.^[2][4] These include: Lys60, Lys132, Arg6, and Arg78.^[1] The are both found on the type 2 binding sites and are entirely buried in the protein; the are partly exposed.^[1] This leaves the interface particularly electropositive; electrostatic repulsion is balanced by several favorable interactions that include the formation of 24 hydrogen bonds, 2 salt bridges and approximately 24% of total hydrophobic surface buried (per subunit).^[4] The trimer is further stabilized by the binding of an oxyanion which would have a neutralizing effect.^[3]

Type 1 binding sites utilize hydrogen bonds and interactions with uncharged polar residues to bind molybdate.^[4] The tight pocket volume and rigidity of the site suggest that there is high selectivity for molybdate (or tungstate because ModG cannot differentiate between the two oxyanions).^[4] Type 2 sites are comparatively larger in pocket volume, supple, and feature electropositive lysine residues that would contribute to high affinity for negatively charged oxyanions like molybdate.^[4] Because the type 2 sites are larger and more flexible, there would be less specificity for molybdate and thus other oxyanions such as phosphate would likely compete for binding.^[3][4]

Function in Bacteria

The ability of A. vinelandii to differentiate between molybdate and other oxyanions is of particular interest to researchers.^[1][2][5] Phosphate is distinguished from molybdate by the divergence in protonation states: phosphate is protonated at physiological pH whereas molybdate is not.^[2] The distinction between sulfate and molybdate is reliant upon differences in ligand size.^[2] Interestingly, molybdate permeases are not able to distinguish between tungstate and molybdate.^[1][2] Regulation of these intracellular metabolites is attributed to other molybdate-binding proteins collectively known as molbindins.^[1][5]

Fate

ModG is eventually degraded and incorporated into molybdopterin, a cofactor of molybdenum enzymes, or the iron-molybdenum cofactor of a nitrogenase enzyme.^[1][2] Currently there is little known about cofactor biosynthesis involving the ModG protein.^[1]