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Monitoring conformational heterogeneity of the lid of DnaK substrate‐binding domain during its chaperone cycle
Author(s) -
Banerjee Rupa,
Jayaraj Gopal Gunanathan,
Peter Joshua Jebakumar,
Kumar Vignesh,
Mapa Koyeli
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13769
Subject(s) - allosteric regulation , förster resonance energy transfer , biophysics , chaperone (clinical) , proteostasis , cyclic nucleotide binding domain , protein structure , chemistry , substrate (aquarium) , peptide , plasma protein binding , nucleotide , biochemistry , biology , crystallography , enzyme , medicine , ecology , physics , pathology , quantum mechanics , gene , fluorescence
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.