A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline |
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Authors: | Tapan Biswas,Olga E. Zolova,Felipe Lombó ,Jose A. Salas,Sylvie Garneau-Tsodikova |
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Affiliation: | 1 Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA 2 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA 3 Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain 4 PharmaMar SA, 28770 Colmenar Viejo, Spain |
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Abstract: | Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 Å resolution reveals that TioX protomer shares an ancient βαβββ fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion. |
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Keywords: | 3HQA, 3-hydroxyquinaldic acid MAD, multiwavelength anomalous dispersion MIR, multiple isomorphous replacement SeMet, selenomethionine QA, quinaldic acid FPLC, fast protein liquid chromatography NHis, N-terminal His6 tagged MM, minimal media |
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