Protein metalation in a nutshell

Abstract

Metalation, the acquisition of metals by proteins, must avoid mis-metalation with tighter binding metals. This is illustrated by four selected proteins that require different metals: all show similar ranked orders of affinity for bioavailable metals, as described in a universal affinity series (the Irving-Williams series). Crucially, cellular protein metalation occurs in competition with other metal binding sites. The strength of this competition defines the intracellular availability of each metal: its magnitude has been estimated by calibrating a cells' set of DNA-binding, metal-sensing, transcriptional regulators. This has established that metal availabilities (as free energies for forming metal complexes) are maintained to the inverse of the universal series. The tightest binding metals are least available. With these availabilities, correct metalation is achieved.

Keywords: Irving-Williams series; cobalt; copper; iron; magnesium; manganese; metal sensor; metalation; nickel; zinc.

Fig. 1

Metal binding to four proteins exemplifies the risk of mis‐metalation. Tightness of binding to the four proteins is shown for the available and exchangeable forms of metals in the cytosol (or Cu^II for periplasmic MncA). Values (black circles) are free energies for forming complexes calculated via the standard relationship ∆G = −RTlnK _A (∆G free energy change, R molar gas constant, T temperature in kelvin, K _A association constant). Note that values are logarithmically related to binding constants. The more negative the value the tighter the binding. Arrows indicate values that were at the minimum or maximum limits of the respective determinations of metal affinity. CbiK is a Co^II chelatase for vitamin B₁₂ biosynthesis [3], Mg^IIGTP‐CobW is a Co^II metallochaperone from an alternative vitamin B₁₂ biosynthetic pathway, Mg^IIGTP‐YeiR is analogous to Mg^IIGTP‐CobW but implicated in handling Zn^II [5], MncA is a Mn^II cupin [4]. Values for MncA are assigned based upon competition between metals (details noted in the text). Insets show percentage occupancies with copper (black) and Zn^II (grey) as a proportion of total metal occupancy. The correct metals are not the tightest binding metals and the orders of binding tend to follow the Irving–Williams series [6, 7].

Fig. 2

Metal availability is the inverse of the Irving–Williams series and decodes correct metalation. Grey bars show the ranges from 10% to 90% of the transcriptional responses of the cognate sensors for each metal as free energies for forming complexes that would be 50% saturated at the respective availability [3]. Metal availability is inverse to the Irving–Williams series. The ranges indicate the range of strengths of competition from exchangeable cytosolic binding sites against which the sensors have evolved to compete to sustain optimal metal availabilities. Metalation of other proteins similarly involves competition with these exchangeable metal binding sites. Black circles and arrows replicate metal binding data from Fig. 1, except for limits to Cu^I binding to MncA where the weakest value is derived from a competition experiment and the tightest inferred to give negligible (1%) Cu^I occupancy. The four cognate metals become apparent when binding is considered in relation to availability, as shown in the insets with Co^II (salmon red), Zn^II (grey), Mn^II (pink) [3, 5]. These proportional metal occupancies are calculated for idealised cells in which the sensors are at the mid‐points of their ranges. Total calculated metal occupancies are 16% CbiK, 99% Mg^IIGTP‐CobW, 36% Mg^IIGTP‐YeiR and (using the selected K _A Mn^II) 91% MncA, implying substantial amounts of apo‐CbiK and apo‐YeiR exist under these conditions. Online metalation calculators similarly decode metal occupancies in the context of defined metal availabilities (https://mib‐nibb.webspace.durham.ac.uk/metalation‐calculators/) [53].