Structural Basis for the Metal-Selective Activation of the Manganese Transport Regulator of Bacillus subtilis^,

Abstract

The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn(2+) to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR.Mn(2+) structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 A (herein designated the AB conformer). Analysis of four additional crystal forms of MntR.Mn(2+) reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or pH 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 A, thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 A. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 A internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn(2+), Cd(2+), and Zn(2+) binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR.

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR

“…This ratio indicates a cooperative binding process which is perhaps better described by the overall equilibrium for the binding of two Mn 2+ ions (2Mn 2+ + apoMntR ↔Mn 2 -MntR) with an equilibrium constant K = K 1 K 2 . Our dissociation constants (K d1 = 900 μM, K d2 = 30 μM, ) are comparable to those determined previously from a AntR (K d1 = 210 μM and K d2 = 17 μM, ) (18) but significantly greater than those obtained from ITC measurements (K d1 = 10 μM and K d2 = 1 μM, ) (15). The value of is more certain than the individual values [radical1] and are also consistent with the results of the ANS and Mag-fura-2 experiments reported here.…”

“…Differences in the buffer conditions for the ITC versus the experiments reported here (500 mM NaCl, pH 8, 10% for ITC vs 200 or 300 mM NaCl, pH 7.2, 5% glycerol) seem inadequate to explain the disparate findings from these two studies. The ITC experiments were reported in large part to confirm the metal/protein monomer binding stoichiometry (2:1 for Mn 2+ and Cd 2+ , 1:1 for Zn 2+ ) observed crystallographically (15). On the basis of the aforementioned discrepancies, we propose that the ITC values are a useful gauge of binding stoichiometry but that the Mag-fura-2 and EPR studies presented here are a more reliable determination of the binding constants.…”

“…Two new reports describe some metal-binding affinities of MntR and a homolog AntR (15,18); our results are compared with these recent findings below. Measuring the metalbinding affinities is an essential component for understanding how MntR selectively responds to its cognate metal ion and elucidating its mechanism of action.…”

“…The AC form of MntR has been observed upon reconstitution with Ca 2+ , Mn 2+ , and Cd 2+ , while the AB form has only been observed with Mn 2+ (13,15). With Mn 2+ , formation of the AB versus AC form is dependent upon conditions such as temperature and pH; from these studies, the AC form has been proposed as the more physiologically relevant conformer (15). The mononuclear A only site form has been observed with Co 2+ (A. Glasfeld, personal communication) and Zn 2+ (15).…”

“…With Mn 2+ , formation of the AB versus AC form is dependent upon conditions such as temperature and pH; from these studies, the AC form has been proposed as the more physiologically relevant conformer (15). The mononuclear A only site form has been observed with Co 2+ (A. Glasfeld, personal communication) and Zn 2+ (15). On the basis of the observation that the A site is conserved in all three structure types, it has been suggested that the A site serves as an "activation filter," and that the formation of a binuclear site is essential for full activation of the protein (15).…”