title: Structural insights into substrate binding by the molecular chaperone DnaK.
abstract: How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.
authors.affiliation: Biophysics Research Division University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109 USA.
authors.forename: M
authors.initials: M
authors.lastname: Pellecchia
authors.forename: D L
authors.initials: DL
authors.lastname: Montgomery
authors.forename: S Y
authors.initials: SY
authors.lastname: Stevens
authors.forename: C W
authors.initials: CW
authors.lastname: Vander Kooi
authors.forename: H P
authors.initials: HP
authors.lastname: Feng
authors.forename: L M
authors.initials: LM
authors.lastname: Gierasch
authors.forename: E R
authors.initials: ER
authors.lastname: Zuiderweg
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli