Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiation of sporulation.

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and microESI-MS. NMR studies show that the divalent metals Ca(2+), Mg(2+) and Mn(2+) primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu(2+) show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. microESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu(2+), multiple metal-bound species are observed. Subsequent experiments reveal that Mg(2+) supports phosphotransfer between KinA and Spo0F, while Cu(2+) fails to support KinA phosphotransfer. Additionally, the presence of Cu(2+) at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.