title: Structure and mechanistic insights into novel iron-mediated moonlighting functions of human J-protein cochaperone, Dph4.
abstract: J-proteins are obligate cochaperones of Hsp70s and stimulate their ATPase activity via the J-domain. Although the functions of J-proteins have been well understood in the context of Hsp70s, their additional co-evolved "physiological functions" are still elusive. We report here the solution structure and mechanism of novel iron-mediated functional roles of human Dph4, a type III J-protein playing a vital role in diphthamide biosynthesis and normal development. The NMR structure of Dph4 reveals two domains: a conserved J-domain and a CSL-domain connected via a flexible linker-helix. The linker-helix modulates the conformational flexibility between the two domains, regulating thereby the protein function. Dph4 exhibits a unique ability to bind iron in tetrahedral coordination geometry through cysteines of its CSL-domain. The oxidized Fe-Dph4 shows characteristic UV-visible and electron paramagnetic resonance spectral properties similar to rubredoxins. Iron-bound Dph4 (Fe-Dph4) also undergoes oligomerization, thus potentially functioning as a transient "iron storage protein," thereby regulating the intracellular iron homeostasis. Remarkably, Fe-Dph4 exhibits vital redox and electron carrier activity, which is critical for important metabolic reactions, including diphthamide biosynthesis. Further, we observed that Fe-Dph4 is conformationally better poised to perform Hsp70-dependent functions, thus underlining the significance of iron binding in Dph4. Yeast Jjj3, a functional ortholog of human Dph4 also shows a similar iron-binding property, indicating the conserved nature of iron sequestration across species. Taken together, our findings provide invaluable evidence in favor of additional co-evolved specialized functions of J-proteins, previously not well appreciated.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Anushikha
authors.initials: A
authors.lastname: Thakur
authors.forename: Balasubramanyam
authors.initials: B
authors.lastname: Chitoor
authors.forename: Arvind V
authors.initials: AV
authors.lastname: Goswami
authors.forename: Gautam
authors.initials: G
authors.lastname: Pareek
authors.forename: Hanudatta S
authors.initials: HS
authors.lastname: Atreya
authors.forename: Patrick
authors.initials: P
authors.lastname: D'Silva
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism; D019143:Evolution, Molecular; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000378:metabolism; D016335:Zinc Fingers / Q000502:physiology
chemical_list: C572774:DNAJC24 protein, human; D004167:Diphtheria Toxin; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D007501:Iron; D000251:Adenosine Triphosphatases