Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a β-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.
P. K. and T. J. were supported by the Heidelberg Biosciences International Graduate School (HBIGS). This work was supported by a grant of the Deutsche Forschungsgemeinschaft ( MO970/7-1 ) to A. M. C. L. received funding from the National Research Foundation of Korea (NRF) funded by the Korea government ( MSIT ) (grant 2021R1C1C1011690 and RS-2023-00217595 ), the Core Research Institute Basic Science Research Program through the NRF funded by the Ministry of Education (grant 2021R1A6A1A10044950 ), and the new faculty research fund of Ajou University .We thank Christian Scholz (Institut für Geowissenschaften, University of Heidelberg) for performing ICP-OES measurements. Janosch Hennig gratefully acknowledges support from the European Molecular Biology Laboratory. P. K. J. H. and A. M. conceptualization; P. K. B. S. J. H. and A. M. methodology; P. K. B. S. S. M. C. L. J. H. and A. M. investigation; P. K. B. S. T. J. S. M. C. L. J. H. and A. M. formal analysis; A. M. and P. K. resources; A. M. writing–original draft; P. K. B. S. T. J. C. L. and J. H. writing–review and editing; A. M. supervision; P. K. and A. M. visualization; C. L. and A. M. funding acquisition. P. K. and T. J. were supported by the Heidelberg Biosciences International Graduate School (HBIGS). This work was supported by a grant of the Deutsche Forschungsgemeinschaft (MO970/7-1) to A. M. C. L. received funding from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (grant 2021R1C1C1011690 and RS-2023-00217595), the Core Research Institute Basic Science Research Program through the NRF funded by the Ministry of Education (grant 2021R1A6A1A10044950), and the new faculty research fund of Ajou University.We thank Christian Scholz (Institut für Geowissenschaften, University of Heidelberg) for performing ICP-OES measurements. Janosch Hennig gratefully acknowledges support from the European Molecular Biology Laboratory .
