title: Semi-Empirical Structure Determination of Escherichia coli Hsp33 and Identification of Dynamic Regulatory Elements for the Activation Process.
abstract: The activation process of the redox-regulated chaperone heat shock protein 33 (Hsp33) is constituted by the oxidation-induced unfolding of the C-terminal zinc-binding domain and concomitant oligomerization of the N-terminal core domain. Herein, the semi-empirical solution structure of Escherichia coli Hsp33 in the reduced, inactive form was generated through conformational space annealing calculations, utilizing minimalistic NMR data and multiple homology restraints. The various conformations of oxidized Hsp33 and some mutant forms were also investigated in solution. Interestingly, a specific region concentrated around the interdomain linker stretch and its interacting counterparts, the N-terminal β-strand 1 and α-helix 1, hardly showed up as signals in the NMR measurements. The NMR spectra of an Hsp33 derivative with a six-residue deletion in the disordered N-terminus implied a plausible conformational exchange associated with the identified region, and the corresponding exchange rate appeared slower than that of the wild type. Subsequent mutations that destroyed the structure of the β1 or α1 elements resulted in the formation of a reduced but active monomer, without the unfolding of the zinc-binding domain. Collectively, structural insights into the inactive and active conformations, including wild-type and mutant proteins, suggest that the dynamic interactions of the N-terminal segments with their contacting counterpart, the interdomain linker stretch, in the reduced, inactive state are the structural determinants regulating the activation process of the post-translationally regulated chaperone, Hsp33.
authors.affiliation: Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea; Department of Molecular Science and Technology, Ajou University, Suwon, Gyeonggi 16499, Republic of Korea.
authors.forename: Yoo-Sup
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea; Department of Bioinformatics, University of Sciences and Technology, Daejeon 34113, Republic of Korea.
authors.forename: Jinhyuk
authors.initials: J
authors.lastname: Lee
authors.affiliation: Protein Structure Group, Korea Basic Science Institute, 162 Yeongudanji-ro, Ochang, Chungbuk 28119, Republic of Korea.
authors.forename: Kyoung-Seok
authors.initials: KS
authors.lastname: Ryu
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center, Plant Molecular Biology and Biotechnology Research Center, Research Institute of Natural Science, Gyeongsang National University, Jinju 52828, Republic of Korea.
authors.forename: Yuno
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea.
authors.forename: Tai-Geun
authors.initials: TG
authors.lastname: Jung
authors.affiliation: Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea.
authors.forename: Jeong-Hwa
authors.initials: JH
authors.lastname: Jang
authors.affiliation: Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea.
authors.forename: Dae-Won
authors.initials: DW
authors.lastname: Sim
authors.affiliation: Protein Structure Group, Korea Basic Science Institute, 162 Yeongudanji-ro, Ochang, Chungbuk 28119, Republic of Korea.
authors.forename: Eun-Hee
authors.initials: EH
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, Gyeonggi 16499, Republic of Korea; College of Pharmacy, Ajou University, Suwon, Gyeonggi 16499, Republic of Korea.
authors.forename: Min-Duk
authors.initials: MD
authors.lastname: Seo
authors.affiliation: Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center, Plant Molecular Biology and Biotechnology Research Center, Research Institute of Natural Science, Gyeongsang National University, Jinju 52828, Republic of Korea.
authors.forename: Keun Woo
authors.initials: KW
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Research Institute (RIBHS) and College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea. Electronic address: wonhs@kku.ac.kr.
authors.forename: Hyung-Sik
authors.initials: HS
authors.lastname: Won
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary
chemical_list: D029968:Escherichia coli Proteins; C492165:HSP33 protein, E coli; D006360:Heat-Shock Proteins
keywords: conformational change; molecular chaperone; nuclear magnetic resonance; oxidative stress; redox regulation