Structural Rearrangement Accompanying Ligand Binding in the GAF Domain of CodY from Bacillus subtilis

TY - JOUR

T1 - Structural Rearrangement Accompanying Ligand Binding in the GAF Domain of CodY from Bacillus subtilis

AU - Levdikov, Vladimir M.

AU - Blagova, Elena

AU - Colledge, Vicki L.

AU - Lebedev, Andrey A.

AU - Williamson, David C.

AU - Sonenshein, Abraham L.

AU - Wilkinson, Anthony J.

PY - 2009/7/31

Y1 - 2009/7/31

N2 - The GAT domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of p-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAT domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure. Descriptions of binding are presently limited by the absence of a GAT domain for which both liganded and unliganded structures are known. Earlier, we solved the crystal structure of the GAF domain of CodY, a branched-chain amino acid and GTP-responsive regulator of the transcription of stationary-phase and virulence genes in Bacillus, in complexes with isoleucine and valine. Here, we report the structure of this domain in its unliganded form, allowing definition of the structural changes accompanying ligand binding. The core of the protein and its dimerisation interface are essentially unchanged, in agreement with circular dichroism spectroscopy experiments that show that the secondary structure composition is unperturbed by ligand binding. There is however extensive refolding of the binding site loops, with up to 15-angstrom movements of the coiled segment linking beta 3 and beta 4, such that the binding pocket is not formed in the absence of the ligand. The implications of these structural rearrangements for ligand affinity and specificity are discussed. Finally, saturation-transfer-difference NMR spectroscopy showed binding of isoleucine but not that of GTP to the GAF domain, suggesting that the two cofactors do not have a common binding site. (C) 2009 Elsevier Ltd. All rights reserved.

AB - The GAT domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of p-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAT domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure. Descriptions of binding are presently limited by the absence of a GAT domain for which both liganded and unliganded structures are known. Earlier, we solved the crystal structure of the GAF domain of CodY, a branched-chain amino acid and GTP-responsive regulator of the transcription of stationary-phase and virulence genes in Bacillus, in complexes with isoleucine and valine. Here, we report the structure of this domain in its unliganded form, allowing definition of the structural changes accompanying ligand binding. The core of the protein and its dimerisation interface are essentially unchanged, in agreement with circular dichroism spectroscopy experiments that show that the secondary structure composition is unperturbed by ligand binding. There is however extensive refolding of the binding site loops, with up to 15-angstrom movements of the coiled segment linking beta 3 and beta 4, such that the binding pocket is not formed in the absence of the ligand. The implications of these structural rearrangements for ligand affinity and specificity are discussed. Finally, saturation-transfer-difference NMR spectroscopy showed binding of isoleucine but not that of GTP to the GAF domain, suggesting that the two cofactors do not have a common binding site. (C) 2009 Elsevier Ltd. All rights reserved.

KW - GAF domain

KW - conformational change

KW - transcription regulation

KW - Bacillus subtilis

KW - branched-chain amino acids

KW - DEPENDENT CONFORMATIONAL-CHANGE

KW - GRAM-POSITIVE BACTERIA

KW - CRYSTAL-STRUCTURE

KW - STATIONARY-PHASE

KW - CGMP BINDING

KW - MACROMOLECULAR STRUCTURES

KW - MOLECULAR-GRAPHICS

KW - GLOBAL REGULATOR

KW - GTP

KW - DIMERIZATION

UR - http://www.scopus.com/inward/record.url?scp=67649873098&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2009.05.077

DO - 10.1016/j.jmb.2009.05.077

M3 - Article

SN - 0022-2836

VL - 390

SP - 1007

EP - 1018

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 5

ER -