title: Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.
abstract: DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures.
authors.affiliation: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.
authors.forename: Rupa
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.|Academy of Scientific and Innovative Research (AcSir), CSIR-CRRI, New Delhi, India.
authors.forename: Gopal Gunanathan
authors.initials: GG
authors.lastname: Jayaraj
authors.affiliation: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.
authors.forename: Joshua Jebakumar
authors.initials: JJ
authors.lastname: Peter
authors.affiliation: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.|Academy of Scientific and Innovative Research (AcSir), CSIR-CRRI, New Delhi, India.
authors.forename: Vignesh
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.|Academy of Scientific and Innovative Research (AcSir), CSIR-CRRI, New Delhi, India.|Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Greater Noida, India.
authors.forename: Koyeli
authors.initials: K
authors.lastname: Mapa
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs
chemical_list: D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli; D003545:Cysteine
keywords: Hsp70; domain allostery; heat shock proteins; proteostasis; single-molecule FRET