Ku-Sung Jo^1,†, Ji-Hun Kim², Kyoung-Seok Ryu³, Joo-Seong Kang¹, Chae-Yeon Wang¹, Yoo-Sup Lee^1,4,5, Min-Duk Seo⁴, Young-Ho Lee³, Hyung-Sik Won^1,*

¹ Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical & Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea
² College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28160, Republic of Korea
³ Protein Structure Group, Korea Basic Science Institute, Ochang, Chungbuk 28199, Republic of Korea
⁴ College of Pharmacy and Department of Molecular Science and Technology, Ajou University, Suwon, Gyeonggi 16499, Republic of Korea
⁵ Present address: Y.-S. Lee, Analytics Team, R&D Division, POLUS Inc., Incheon 21984, Republic of Korea.

^† K.-S. Jo was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant no. G201809G00019).

^*Corresponding author : Hyung-Sik Won

The various chaperone activities of heat shock proteins contribute to ensuring cellular proteostasis. Here, we demonstrate the non-canonical unfoldase activity as an inherent functionality of the prokaryotic molecular chaperone, Hsp33. Hsp33 was originally identified as a holding chaperone that is post-translationally activated by oxidation. However, in this study, we verified that the holding-inactive reduced form of Hsp33 (^RHsp33) strongly bound to the translational elongation factor, EF-Tu. This interaction was critically mediated by the redox-switch domain of ^RHsp33 and the guanine nucleotide-binding domain of EF-Tu. The bound ^RHsp33, without undergoing any conformational change, catalyzed the EF-Tu aggregation by evoking the aberrant folding of EF-Tu to expose hydrophobic surfaces. Consequently, the oligomers/aggregates of EF-Tu, but not its functional monomeric form, were highly susceptible to proteolytic degradation by Lon protease. These findings present a unique example of an ATP-independent molecular chaperone with distinctive dual functions—as an unfoldase/aggregase and as a holding chaperone—depending on the redox status. It is also suggested that the unusual unfoldase/aggregase activity of ^RHsp33 can contribute to cellular proteostasis by dysregulating EF-Tu under heat-stressed conditions.

Abbreviations
ANS, 8-anilino-1-naphthalene sulfonic acid; CD, circular dichroism; D2, domain-II; D3, domain-III; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EF-Ts, elongation factor thermal-stable; EF-Tu, elongation factor thermal-unstable; GD, guanidine nucleotide-binding domain; Hsp33, heat shock protein 33; ITC, isothermal titration calorimetry; MLD, middle linker domain; RSD, redox-switch domain; TROSY, transverse relaxation optimized spectroscopy

Keywords : proteostasis; protein quality control; protein turnover; protein misfolding; protein aggregation