| Autor: |
Papsdorf K; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany.; Stanford University School of Medicine, Department of Genetics, 300 Pasteur Drive, Stanford, CA, 94305, USA., Sima S; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany., Schmauder L; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany., Peter S; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany., Renner L; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany., Hoffelner P; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany., Richter K; Center for integrated protein research at the Department of Chemie, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany. klaus.richter@richterlab.de. |
| Abstrakt: |
The molecular chaperone Hsc70 performs essential tasks by folding proteins. Hsc70 is driven by the hydrolysis of ATP and tuned by the association with various co-chaperones. One such cofactor is the nematode nucleotide exchange factor UNC-23, whose mutation disrupts muscle attachment and induces a severe head-bent phenotype in C.elegans. Interestingly, four mutations in Hsc70 can suppress this phenotype, but the molecular mechanism underlying this suppression is unknown. Here we characterize these four suppressor variants, Hsc70 D233N, S321F, A379V and D384N. In vitro only Hsc70 S321F shows reduced stability and altered nucleotide interaction, but all mutations affect the ATPase stimulation. In particular, Hsc70 D233N and Hsc70 A379V show strongly reduced interactions with DNJ-12 and DNJ-13. Nucleotide exchange factor binding instead is barely influenced in Hsc70 D233N, A379V and D384N and their chaperone activity is preserved. Molecular dynamics simulations suggest that effects in Hsc70 S321F and Hsc70 A379V originate from steric clashes in the vicinity of the mutation site, while D233N disrupts a salt bridge that contributes to Hsc70's nucleotide-induced conformational changes. In summary, the analyzed mutants show altered ATPase and refolding activity caused by changes in Hsp40 binding. |
