Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Teplyakov, A.; Obmolova, G.; Wilson, K.S.; Kuromizu, Kenji

doi:10.1111/j.1432-1033.1993.tb17814.x

Cited by 41 publications

(39 citation statements)

References 43 publications

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“…To examine the static structural change with binding of the chromophore, we compared holo-and apo-NCS in crystalline coordinates of the Protein Data Bank (20,50). However, there is very little conformational change, even for the loop structures (20).…”

Section: Resultsmentioning

confidence: 99%

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Takashima

Yoshida

Ishino

et al. 2005

Journal of Biological Chemistry

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Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulinlike seven-stranded anti-parallel ␤-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13 C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the ␤-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.Holo-neocarzinostatin (holo-NCS) 1 (molecular mass 12 kDa, 113 amino acid residues ϩ chromophore) is a prominent member of the strongest anti-tumor reagent chromoprotein family (Fig. 1, B and D) (1). It is a non-covalent complex of an enediyne ring chromophore and its carrier protein isolated from Streptomyces carzinostaticus (2-4). The chromophore, which has a selective bulged DNA-cleaving activity (5), is easily inactivated by light, heat, and molecular oxygen because of its labile structure. Despite its intrinsic instability in the free state, the chromophore in the complex is stable even in vivo. It is thought that holo-NCS penetrates into the target cell carrying the chromophore and promptly releases it at the cell nucleus to kill the cell. Therefore, the holo-NCS family has attracted much attention in clinical use as well as for use in targeting drug delivery systems (6 -14). The three-dimensional structures of holo-NCS and the chromoprotein family have been investigated by solution NMR (12, 15-19) and x-ray diffraction (20), elucidating an immunoglobulin-like sevenstranded anti-parallel ␤-barrel structure (Fig. 1B) (21).Based on the previous NMR and x-ray studies for holo-NCS, several mechanisms of the chromophore stabilization have been proposed. These mechanisms were dominated by hydrophobic interactions with the binding pocket. However, the mechanism of releasing and binding of the chromophore remains unclear, even though the process has attracted much atte...

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Section: Resultsmentioning

confidence: 99%

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Takashima

Yoshida

Ishino

et al. 2005

Journal of Biological Chemistry

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“…Because backbone conformation of apoNCS and holoNCS is superimposable [22,23], it is conceivable that conformational changes do not contribute to the enhanced stability of the holoNCS protein. …”

Section: Ncs-c(b) Contributes To the Enhanced Stability Of Holoncsmentioning

confidence: 99%

“…1 illustrates the holoNCS model in a neutral aqueous environment [21]. NCS has a superimposable backbone three-dimensional structure in both its holo and apo forms [22,23]. NCS-C is highly sensitive to chemicals, basic pH, light, and heat [24].…”

mentioning

confidence: 99%

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Jayachithra¹,

Hariharan²,

Kumar³

et al. 2008

Analytical Biochemistry

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Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the proteinbound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability. KeywordsEnediyne; Neocarzinostatin; Chromophore; Ligand binding; Holoprotein; Protein stability Recently, much attention has been focused on the protein-ligand conjugation. Most conjugate proteins undergo changes in their conformation and subsequently, in stability on ligand removal [1][2][3]. For example, binding of heme to cytochrome changes the protein conformation and consequently, increases the protein stability [4]. It is rather uncommon for conjugate proteins to adopt a like conformation with or without the ligand. This includes azurin (β protein with a copper ion), flavodoxin (α/β protein with a flavin mononucleotide) [5], neocarzinostatin (NCS, 1 β protein with an enediyne chromophore) [6,7] and so on. In spite of structural similarity, the holo form of azurin is more stable than its apo form [8]. The flavodoxin holo form, in contrast, has little changes in either conformation or stability on its cofactor removal *Corresponding author. Fax: +886 4 22862547. E-mail address: chdhchin@dragon.nchu.edu.tw (D.-H. Chin). 1 Abbreviations used: NCS, neocarzinostatin; holoNCS, neocarzinostatin chromoprotein conjugate complex; apoNCS, apoprotein of neocarzinostatin; NCS-C, neocarzinostatin chromophore; NCS-C(b), protein-bound neocarzinostatin chromophore; CD, circular dichroism; HPLC, high-performance liquid chromatography; EtBr, ethidium bromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; T m , temperature at which half of the protein is unfolded; T r , temperature at which half of the NCS-C(b) is released from NCS complex; C m , concentration of denaturant at which half of the protein is unfolded; C r , concentration of denaturant at which half of the NCS-C(b) is released from NCS com...

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“…[25,28] The three-dimensional holo-NCS structure was downloaded from the Brookhaven data bank (file pdb.1nco.ent), which had been determined from crystals that were grown in a 2-methyl-2,4-pentanediol solution (70 %) under acidic pH. With a help of a molecular modeling simulation program, the acidified solid-state form was used as the basis to simulate the form expected under aqueous conditions and neutral pH.…”

Section: Effect Of the Chemical Functional Groups Attached To The Thiolmentioning

confidence: 99%

Rejection by Neocarzinostatin Protein through Charges Rather than Sizes

Chin¹

1999

Chem. Eur. J.

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Neocarzinostatin is a very potent antitumor antibiotic from Streptomyces carzinostaticus. It consists of a carrier apoprotein and an active enediyne chromophore. Thiols can induce irreversible inactivation as a result of cycloaromatization of the enediyne moiety and the protein is responsible for selecting thiols with which to react. While 2-mercaptoethanol can inactivate the protein-bound chromophore, glutathione is rejected by the protein. This selective inactivation has been suggested to be the basis of the self-protective functionality of the microorganisms that produce neocarzinostatin. We have used screening tests with various thiols and investigated the pH-dependency of the reaction and have thus clearly demonstrated that neocarzinostatin apoprotein rejects thiols by means of electrostatic repulsion of negative charges. In contrast to what one would generally predict from the lock-and-key model, we have found that size or shape exclusion does not have major effects. The acidic residues of the apoprotein, in particular, Asp 33, Asp 79, and Asp 99, are located near to the site of thiolate attack. The ionized carboxylate side chains of the amino acid residues repel the negatively charged glutathione by electrostatic forces. Our model resolves the longstanding puzzle of how the neocarzinostatin apoprotein is responsible for shielding the labile chromophore from attack by the most abundant eukaryote cellular thiol before the drug reaches the target DNA.

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Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Cited by 41 publications

References 43 publications

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Rejection by Neocarzinostatin Protein through Charges Rather than Sizes

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