title: Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, fromBacillus subtilis.
abstract: CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases,adenylate cyclases,FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY fromBacillus subtilisrevealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.
authors.affiliation: From the Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom.
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Levdikov
authors.affiliation: From the Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom.
authors.forename: Elena
authors.initials: E
authors.lastname: Blagova
authors.affiliation: From the Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom.
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Young
authors.affiliation: the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Boris R
authors.initials: BR
authors.lastname: Belitsky
authors.affiliation: the STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxfordshire OX11 0QX, United Kingdom.
authors.forename: Andrey
authors.initials: A
authors.lastname: Lebedev
authors.affiliation: the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Abraham L
authors.initials: AL
authors.lastname: Sonenshein
authors.affiliation: From the Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom, tony.wilkinson@york.ac.uk.
authors.forename: Anthony J
authors.identifier: 0000-0003-4577-9479
authors.initials: AJ
authors.lastname: Wilkinson
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D008024:Ligands
keywords: Bacillus; DNA-binding protein; X-ray crystallography; protein structure; transcription repressor