Structural Analysis of Divalent Metals Binding to the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Response Regulator Spo0F: The Possibility for <Emphasis Type="Italic">In Vitro</Emphasis> Metalloregulation
in the Initiation of Sporulation |
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Authors: | Douglas?J?Kojetin Richele?J?Thompson Linda?M?Benson Stephen?Naylor Jenora?Waterman Keith?G?Davies Charles?H?Opperman Keith?Stephenson James?A?Hoch Email author" target="_blank">John?CavanaghEmail author |
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Institution: | (1) Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA;(2) Biomedical Mass Spectrometry and Functional Proteomics Facility, Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA;(3) Department of Plant Pathology, Center for the Biology of Nematode Parasitism, North Carolina State University, Raleigh, NC 27695, USA;(4) Nematode Interaction Unit, Rothamsted Research Limited, Harpenden, Herts, AL52JQ, UK;(5) Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA;(6) Division of Biological Engineering, Room 56-738, MIT, Cambridge, MA, 02139, USA;(7) School of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK |
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Abstract: | 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 μESI-MS. NMR studies show that the divalent metals Ca2+, Mg2+ and Mn2+ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu2+ 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 β4-α4 loop and the β5/α5 interface, particularly around and including H101. μESI-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 Cu2+, multiple metal-bound species are observed. Subsequent experiments reveal that Mg2+ supports phosphotransfer between KinA and Spo0F, while Cu2+ fails to support KinA phosphotransfer. Additionally, the presence of Cu2+ 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. |
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Keywords: | metal binding NMR spectroscopy response regulator sporulation two-component signal transduction |
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