Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.
DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.
Gelinas
Amy D
AD
Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472, USA.
Toth
Joseph
J
Bethoney
Kelley A
KA
Stafford
Walter F
WF
Harrison
Celia J
CJ
D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C063842:dnaK protein, E coli; D000409:Alanine