Contributions of the LPPVK motif of the iron-sulfur template protein IscU to interactions with the Hsc66-Hsc20 chaperone system.

Hsc66 (HscA) and Hsc20 (HscB) from Escherichia coli comprise a specialized chaperone system that selectively binds the iron-sulfur cluster template protein IscU. Hsc66 interacts with peptides corresponding to a discrete region of IscU including residues 99-103 (LPPVK), and a peptide containing residues 98-106 stimulates Hsc66 ATPase activity in a manner similar to IscU. To determine the relative contributions of individual residues in the LPPVK motif to Hsc66 binding and regulation, we have carried out an alanine mutagenesis scan of this motif in the Glu98-Cys106 peptide and the IscU protein. Alanine substitutions in the Glu98-Cys106 peptide resulted in decreased ATPase stimulation (2-10-fold) because of reduced binding affinity, with peptide(P101A) eliciting <10% of the parent peptide stimulation. Alanine substitutions in the IscU protein also revealed lower activities resulting from decreased apparent binding affinity, with the greatest changes in Km observed for the Pro101 (77-fold), Val102 (4-fold), and Lys103 (15-fold) mutants. Calorimetric studies of the binding of IscU mutants to the Hsc66.ADP complex showed that the P101A and K103A mutants also exhibit decreased binding affinity for the ADP-bound state. When ATPase stimulatory activity was assayed in the presence of the co-chaperone Hsc20, each of the mutants displayed enhanced binding affinity, but the P101A and V102A mutants exhibited decreased ability to maximally simulate Hsc66 ATPase. A charge mutant containing the motif sequence of NifU, IscU(V102E), did not bind the ATP or ADP states of Hsc66 but did bind Hsc20 and weakly stimulated Hsc66 ATPase in the presence of the co-chaperone. These results indicate that residues in the LPPVK motif are important for IscU interactions with Hsc66 but not for the ability of Hsc20 to target IscU to Hsc66. The results are discussed in the context of a structural model based on the crystallographic structure of the DnaK peptide-binding domain.