GrpE N-terminal domain contributes to the interaction with Dnak and modulates the dynamics of the chaperone substrate binding domain.
GrpE acts as a nucleotide exchange factor for DnaK, the main Hsp70 protein in bacteria, accelerating ADP/ATP exchange by several orders of magnitude. GrpE is a homodimer, each subunit containing three structural domains: a N-terminal unordered segment, two long coils and a C-terminal globular domain formed by a four-helix bundle, and a beta-subdomain. GrpE association to DnaK nucleotide-binding domain involves side-chain and backbone interactions located within the "headpiece" of the cochaperone, which consists of the C-terminal half of the coils, the four-helix bundle and the beta-subdomain. However, the role of the GrpE N-terminal region in the interaction with DnaK and the activity of the cochaperone remain controversial. In this study we explore the contribution of this domain to the binding reaction, using the wild-type proteins, two deletion mutants of GrpE (GrpE(34-197) and GrpE(69-197)) and the isolated DnaK nucleotide-binding domain. Analysis of the thermodynamic binding parameters obtained by isothermal titration calorimetry shows that both GrpE N-terminal segments, 1-33 and 34-68, contribute to the binding reaction. Partial proteolysis and substrate dissociation kinetics also suggest that the N-terminal half of GrpE coils (residues 34-68) interacts with DnaK interdomain linker, regulates the nucleotide exchange activity of the cochaperone and is required to stabilize DnaK-substrate complexes in the ADP-bound conformation.
Moro
Fernando
F
Unidad de Biofísica (CSIC/UPV-EHU) y Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco, Apartado 644, 48080 Bilbao, Spain. fernando.moro@ehu.es
Taneva
Stefka G
SG
Velázquez-Campoy
Adrián
A
Muga
Arturo
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010206:Papain / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D010206:Papain; C063842:dnaK protein, E coli