Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE.
Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.
From the Centro de Investigaciones Biológicas (CIB-CSIC), 28040 Madrid.
Roberto
R
Melero
the Unidad de Biofísica (CSIC/UPV-EHU) and Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco, 48080 Bilbao, and.
Fernando
F
Moro
the Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain.
María Ángeles
MÁ
Pérez-Calvo
the Unidad de Biofísica (CSIC/UPV-EHU) and Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco, 48080 Bilbao, and.
Judit
J
Perales-Calvo
the Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain.
Lucía
L
Quintana-Gallardo
From the Centro de Investigaciones Biológicas (CIB-CSIC), 28040 Madrid, ollorca@cib.csic.es.
Oscar
O
Llorca
the Unidad de Biofísica (CSIC/UPV-EHU) and Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco, 48080 Bilbao, and arturo.muga@ehu.es.
Arturo
A
Muga
the Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain jmv@cnb.csic.es.
José María
JM
Valpuesta
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000494:Allosteric Regulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli
70-Kilodalton Heat Shock Protein (Hsp70); Chaperone; Chaperone DnaK (DnaK); Electron Microscopy (EM); GrpE; Nucleotide Exchange Factor; Protein Folding