title: A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
abstract: Highly conserved molecular chaperone Hsp70 heat shock proteins play a key role in maintaining protein homeostasis (proteostasis). DnaK, a major Hsp70 in Escherichia coli, has been widely used as a paradigm for studying Hsp70s. In the absence of ATP, purified DnaK forms low-ordered oligomer, whereas ATP binding shifts the equilibrium toward the monomer. Recently, we solved the crystal structure of DnaK in complex with ATP. There are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, the interfaces between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer. Biochemical analyses of DnaK protein supported the formation of dimer in solution. Furthermore, our cross-linking experiment based on the DnaK-ATP structure confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. Four mutations, R56A, T301A, N537A, and D540A, resulted in loss of chaperone activity and compromised the formation of dimer, indicating the functional importance of the dimer. Surprisingly, neither the intrinsic biochemical activities, the ATP-induced allosteric coupling, nor GrpE co-chaperone interaction is affected appreciably in all of the mutations except for R56A. Unexpectedly, the interaction with co-chaperone Hsp40 is significantly compromised. In summary, this study suggests that DnaK forms a transient dimer upon ATP binding, and this dimer is essential for the efficient interaction of DnaK with Hsp40.
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Evans Boateng
authors: EB
authors: Sarbeng
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Qingdai
authors: Q
authors: Liu
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Xueli
authors: X
authors: Tian
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Jiao
authors: J
authors: Yang
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Hongtao
authors: H
authors: Li
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Jennifer Li
authors: JL
authors: Wong
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298.
authors: Lei
authors: L
authors: Zhou
authors: From the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298 qliu3@vcu.edu.
authors: Qinglian
authors: Q
authors: Liu
mesh_terms: D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C063842:dnaK protein, E coli
keywords: 70-Kilodalton Heat Shock Protein (Hsp70); Chaperone DnaJ (DnaJ); Chaperone DnaK (DnaK); Heat Shock Protein (HSP); Hsp40; Protein Folding; Protein-Protein Interaction
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