Protein flexural dysfunction: Loss of structural rigidity in a Hsp40 J‐domain mutant correlates with loss of Hsp70 activation

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Functions of the Hsp40/DnaJ ‐ Hsp70/DnaK chaperone machine involve cyclic conformational changes in DnaK that are coupled to ATP hydrolysis in the ATPase domain of DnaK and stimulated by the J‐domain of DnaJ. Experiments with recombinant forms of the DnaJ J‐domain (Jd) and the DnaK ATPase domain (Kase) reveal that a Jd mutant with a D‐to‐N substitution in the conserved HPD tripeptide (JdD35N), has increased affinity for Kase but is unable to stimulate ATP hydrolysis in DnaK. NMR studies on Jd and JdD35N revealed increased bending of Helix II in JdD35N upon binding to Kase. We hypothesize that J‐domain structural rigidity is essential for its stimulation of ATP hydrolysis. Jd conformational strain was measured by 15N‐1H chemical shift perturbations in heteronuclear single quantum correlation (HSQC) NMR spectra, and Jd‐Kase affinity was modulated by potassium ion concentration. Jd conformational strain increased at higher binding affinity when a mutation in Kase prevented its own conformational change. Comparison of the slopes of integrated chemical shift perturbation versus ΔGb for Jd and JdD35N bound to the Kase mutant indicate decreased rigidity in JdD35N. Further experiments with ATP‐bound Kase indicate that structural rigidity in the J‐domain is required for stabilizing the ATP‐bound conformation of the DnaK ATPase domain and stimulating DnaK function. This work was supported by NSF grant MCB‐0349578.