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. 2014 Oct 17;9(10):2347-58.
doi: 10.1021/cb500327m. Epub 2014 Aug 13.

Structure-guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19

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Free PMC article

Structure-guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19

Sherif I Elshahawi et al. ACS Chem Biol. .
Free PMC article

Abstract

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

Figures

Figure 1
Figure 1
(A) Selected structures of naturally occurring 10-membered enediynes. The “warhead” is highlighted in red. (B) Proposed mechanism of cycloaromatization of 1 and its effect on DNA scission and CalC self-sacrifice mechanism.
Figure 2
Figure 2
Structure of CalU16. (A) Overlay of NMR (green) and monomers of the crystal (brick- red) structures of CalU16. The N- and C-termini of the protein are labeled, while the dynamic loop is colored yellow. (B) Monomer with secondary structural elements labeled. The residues in the hydrophobic cavity are represented as stick models. (C) B-factors for the Cα atoms in the crystal structure (left) and Cα RMSD values from the NMR ensemble (right) are mapped to the color and tube diameter of “putty” traces showing the general agreement (correlation coefficient 0.559) between the structures.
Figure 3
Figure 3
CalU16 structural homologues. (A) CalU16 (PDB: 4FPW); (B) CalC (PDB: 2L65), calicheamicin resistance protein; (C) TcmN Aro/Cyc (in complex with trans-dihydroquercetin; PDB: 3TVQ) involved in the biosynthesis of tetracenomycin; (D) Hyp-1 (in complex with ethylene glycol; PDB: 3IE5) involved in the biosynthesis of hypericin; (E) NCS (in complex with hydroxybenzaldehyde; PDB: 2VQ5) involved in the biosynthesis of norcoclaurine.
Figure 4
Figure 4
CalU16 and CalU19 assays. Serial disc dilutions of 1 against (A) pSE28a-E. coli (control), (B) pSECalU16-E. coli (CalU16), (C) pSECalU19-E. coli (CalU19), (D) pJB2011-E. coli (CalC). Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively. Coomassie-stained 8–12% SDS-PAGE gradient gel of (E) CalU16 and (F) CalU19 in the presence of DTT (lane 1), 1 (lane 2), and DTT and 1 (lane 3).
Figure 5
Figure 5
Site directed mutagenesis of CalU16 and CalU19. Docking models of (A) CalC (B) CalU16, and (C) CalU19 with mutated glycine residues represented as spheres where colored Gly residues indicate cleavage sites, wheat Gly residues indicate mutations that did not affect activity and calicheamicin (1) is represented as a stick model. Results of disc diffusion assay in CalU16 mutants (D) pSE28a-E. coli (control); (E) pSEU16G128V-E. coli; (F) pSEU16G128R-E. coli; (G) pSECalU16-E. coli; and CalU19 mutants (H) pSE28a-E. coli (control); (I) pSEU19G177V-E. coli; (J) pSEU19G177R-E. coli; (K) pSECalU19-E. coli. Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively.

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References

    1. Maiese W. M.; Lechevalier M. P.; Lechevalier H. A.; Korshalla J.; Kuck N.; Fantini A.; Wildey M. J.; Thomas J.; Greenstein M. (1989) Calicheamicins, a novel family of antitumor antibiotics: taxonomy, fermentation and biological properties. J. Antibiot. (Tokyo) 42, 558–563. - PubMed
    1. Lee M. D.; Manning J. K.; Williams D. R.; Kuck N. A.; Testa R. T.; Borders D. B. (1989) Calicheamicins, a novel family of antitumor antibiotics. 3. Isolation, purification and characterization of calicheamicins β 1Br, γ 1Br, α 2I, α 3I, β 1I, γ 1I, and δ 1I. J. Antibiot. (Tokyo) 42, 1070–1087. - PubMed
    1. Lee M. D.; Dunne T. S.; Chang C. C.; Siegel M. M.; Morton G. O.; Ellestad G. A.; McGahren W. J.; Borders D. B. (1992) Calicheamicins, a novel family of antitumor antibiotics. 4. Structure elucidation of calicheamicins β 1Br, γ 1Br, α 2I, α 3I, β 1I, γ 1I, and δ 1I. J. Am. Chem. Soc. 114, 985–997. - PubMed
    1. Thorson J. S.; Sievers E. L.; Ahlert J.; Shepard E.; Whitwam R. E.; Onwueme K. C.; Ruppen M. (2000) Understanding and exploiting nature’s chemical arsenal: The past, present, and future of calicheamicin research. Curr. Pharm. Des. 6, 1841–1879. - PubMed
    1. Galm U.; Hager M. H.; Van Lanen S. G.; Ju J.; Thorson J. S.; Shen B. (2005) Antitumor antibiotics: Bleomycin, enediynes, and mitomycin. Chem. Rev. 105, 739–758. - PubMed

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