A superior drug carrier – aponeocarzinostatin in partially unfolded state fully protects the labile antitumor enediyne
© Shanmuganathan et al; licensee BioMed Central Ltd. 2009
Received: 18 February 2009
Accepted: 23 May 2009
Published: 23 May 2009
Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier.
We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy.
The partially unfolded protein is in molten globule-like state, in which ~60% and ~20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state.
These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.
Neocarzinostatin (NCS) is the most studied member within the family of natural enediyne-based chromoproteins with potent anti-tumor activity [1, 2]. Holoneocarzinostatin (holoNCS) drug consists of a biologically active chromophore (NCS-Chr) that is non-covalently bound to a carrier apoprotein (apoNCS). NCS-Chr is very labile and can be inactivated quickly when it is not associated with apoNCS [1, 3, 4]. To carry out the protection role, a regular drug carrier protein must fold properly to form a well-defined specific binding cleft before it can accommodate the ligand molecule. Here we report an interesting observation that apoNCS in its partially unfolded intermediate state is able to efficiently bind and protect the labile NCS-Chr. Elucidation of the protein folding with respect to chromophore protection could serve as a starting point for rational drug carrier design strategies.
Structural characterization of intermediates that populate in the folding/unfolding process is crucial to understand the protein folding mechanism. Equilibrium and kinetic intermediates have been identified in the unfolding/refolding reactions of several proteins [12–15]. The best studied intermediate is the molten globule (MG) state [16, 17]. The MG states are believed to be general folding intermediates because they populate both in the equilibrium and kinetic folding/unfolding pathways [16, 17]. In the present study, we identified and characterized a stable intermediate in the guanidine hydrochloride (GdnHCl)-induced equilibrium unfolding pathway of apoNCS. Intermediate accumulates maximally in 1.2 M GdnHCl under acidic condition and has structural properties resembling that of a MG-like state. To gain insights into the biological role of apoNCS as a drug carrier, the interaction of MG-like intermediate with NCS-Chr was investigated.
NCS powder consisting apoNCS and NCS-Chr in a 1:1 molar ratio was a gift from Kayaku Co., Ltd., Itabashi-Ku, Tokyo, Japan. Extracted NCS-Chr was obtained after repeated methanol extractions of lyophilized NCS stock (0.5 mM, as determined by ε340 = 10,800 M-1 cm-1) in 20 mM sodium citrate (pH 4), following the previously described method . Extracted NCS-Chr was stored at -80°C in amber glass vials. Integrity and concentration of NCS-Chr were examined by UV spectroscopy and HPLC analysis. Labeled 15NH4Cl and D2O were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All other chemicals used were of high quality analytical grade. All experiments were performed in 10 mM phosphate buffer (pH 3) at 25°C.
Expression and purification of apoNCS
Recombinant apoNCS was overexpressed and purified by the procedure as described . Homogeneity of the protein was examined by UV, HPLC and SDS-PAGE. Protein yield was about 2 mg/L. Molecular mass of the purified protein was verified using ESI-Mass analysis.
Preparation of isotope-enriched apoNCS
Uniform 15N labeling was achieved by the established procedure . E. coli BL21 Codon Plus strain (Stratagene, La Jolla, CA, USA) carrying the apoNCS gene was cultured in M9 minimal medium containing 15NH4Cl supplemented with vitamin B1. Final yield of the 15N-labeled protein was about half of the corresponding unlabeled protein expressed in LB medium.
Steady-State fluorescence measurements
Fluorescence spectra were collected using a Hitachi F-4500 spectrofluorimeter at a resolution of 2.5 or 10 nm. For GdnHCl-induced unfolding study, excitation wavelength was set at 280 nm. Intrinsic fluorescence measurements were made at 25°C. Binding affinity of 1-anilino-8-napthalene sulfonate (ANS) to apoNCS at various concentrations of GdnHCl was monitored in a wavelength range of 375 to 625 nm using an excitation wavelength at 355 nm. Excitation and emission bandwidths were set at 5 nm. Concentration of ANS and protein was 100 μM and 10 μM respectively. All samples were prepared in 10 mM phosphate buffer at pH 3.
Circular dichroism (CD) spectroscopy
All CD measurements are carried out on a Jasco J-715 spectropolarimeter (Tokyo, Japan) equipped with a circulating water bath (Neslab, model RTE-140) (Portsmouth, NH, USA). Measurements were made using a 0.1 cm path-length water-jacketed quartz cell. Each spectrum represents an average of 30 scans with a scan speed of 50 nm/min. Concentration of the protein used was 15 μM. Background corrections were made in all spectra. Bandwidth was set to 1 nm and all spectra were acquired at 25°C.
Size-exclusion chromatography (SEC)
Gel-filtration experiments were carried out at 25°C on a superdex-100 column using a Pharmacia AKTA FPLC chromatographic device (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Column was equilibrated with 2 bed volumes of 10 mM phosphate buffer (pH 3) containing appropriate concentrations of GdnHCl at a flow rate of 1 ml/min. Concentration of protein used for each analysis was approximately 250 μg/ml (dissolved in appropriate concentrations of GdnHCl). Protein elution was monitored by UV absorbance at 280 nm.
Thermal denaturation experiments
Thermal stability of apoNCS in 10 mM phosphate buffer (pH 3) in the presence or absence of 1.2 M GdnHCl was monitored by far-UV CD at 224 nm. Changes in ellipticity with temperature were followed from 5–91°C at an increment of 3°C. Experiments were performed using a water-jacketed cell connected to a thermal circulator equipped with a microprocessor and temperature sensor. Protein sample (25 μM) was allowed to equilibrate for 10 minutes at each temperature before data acquisition.
The NMR experiments were carried out on a Bruker DMX 600 MHz NMR spectrometer (Rheinstetten, Germany) at 25°C. A 5 mm inverse probe with a self-shielded z-gradient was used to obtain all gradient-enhanced 1H-15N HSQC spectra [20, 21]. 15N decoupling during acquisitions was achieved using the GARP sequence . Total of 2048 complex data points were collected in the 1H-dimension of the 1H-15N HSQC experiments. In the indirect 15N-dimension spectra, 512 complex data points were collected. The HSQC spectra were recorded by 32 scans at all concentrations of GdnHCl. 15N chemical shifts were referenced using consensus ratio of 0.0101329118. All spectra were processed on a Silicon Graphics workstation using XWINNMR and Sparky softwares.
Binding experiments were performed in 10 mM phosphate buffer (pH 3) by incubating 20 μM apoNCS (in the presence or absence of 1.2 M GdnHCl) with NCS-Chr at 1:1 molar ratio for 30 minutes at 25°C. The final methanol content introduced from NCS-Chr stock was kept minimal to about 4% (v/v). Analyses of the protein-bound NCS-Chr after binding experiments were performed through a Waters μ-Bondapak reverse phase C18 column by a Waters Millennium HPLC equipped with a model 600E solvent delivery system, a 996 photodiode array detector and either a Waters 474 or a Jasco FP-1520 fluorescence detector following previously described method .
GdnHCl-induced unfolding of apoNCS does not follow a two-state model
A stable equilibrium unfolding intermediate of apoNCS
Partially unfolded apoNCS intermediate resembles MG-like state
Thermal stability of MG-like intermediate of apoNCS
NMR studies on structural changes in MG-like state of apoNCS
ApoNCS in partially unfolded MG-like state fully protects labile NCS-Chr
Protection of NCS-Chr against degradation by binding with MG-like state of apoNCS
Remaining % of NCS-Chr a
NCS-Chr + 1.2 M GdnHCl
13 ± 4% b
holoNCS (NCS-Chr + 1:1 folded state of apoNCS)
100 ± 4%
NCS-Chr + 1:1 MG-like state of apoNCS (in 1.2 M GdnHCl)
101 ± 4% c
MG-like state of apoNCS resumes its folded state after binding to NCS-Chr
Understanding the mechanism by which a protein folds from denatured state into its unique native three-dimensional structure is an important problem in molecular biology [33, 34]. ApoNCS, being a model for small all β-sheet proteins, has been extensively investigated for its folding/unfolding pathways [6–11]. Formation of intermediate state has been suggested when apoNCS is treated with 45% trifluoroethanol at pH 5 . Under aqueous condition, we demonstrated that the thermal unfolding of apoNCS at pH 7 follows a two-state mechanism . On the contrary, small-angle neutron scattering studies on apoNCS at pH 7 reveal that there could be several discrete intermediate species at equilibrium populated in the unfolding pathways [9, 10]. The distribution of those substates shows various degrees of residual structures and appears to be temperature or solvent dependent. The major species present during the transition could be considerably unstructured, which may make the thermal unfolding transition look like a two-state transition . This accounts for the failure of identifying a well-defined intermediate state in the unfolding pathway of apoNCS at pH 7 . In the present study, we identified and characterized a stable MG-like state in the GdnHCl-induced unfolding pathway of apoNCS only at a rather acidic pH (pH 3). At pH 5, where apoNCS is stable at room temperature , we have shown that apoNCS is highly resistant against denaturants . The transitions of apoNCS induced by GdnHCl can not be complete even at the highest concentration of the denaturant. Here we also screened GdnHCl-induced unfolding pathways of aqueous apoNCS at 25°C over a pH range of 3 to 9 (data not shown). We could not observe any stable intermediates except at pH 3. Conceivably, the chances of characterizing a stable intermediate are limited by conditions. We wish the present characterization of a MG-like state intermediate would provide some inputs for further understanding of the complex folding/unfolding mechanism of apoNCS.
Potent anti-neoplastic activity of NCS comes from its NCS-Chr, and apoNCS serves its functional role as a carrier and protector [1, 3, 4]. Without apoNCS, NCS-Chr is very labile and can be inactivated quickly by bases, light, heat, and chemicals such as cellular thiols [1, 3, 4]. When NCS-Chr and apoNCS are biosynthesized from specific gene clusters that produce NCS, the peptide chain of apoNCS needs to fold properly and efficiently to form specific binding cleft for accommodation of the chromophore. Statistical studies have suggested that proteins with more complex topologies such as β-sheets usually fold more slowly than proteins with α-helices . ApoNCS being an all β-sheet protein, its topology is not favorable for fast folding. Although the role of ligand in protein folding is not well understood, there are studies showing that binding of a ligand prior to protein folding can significantly accelerate the formation of functional protein . Our in vitro experimental data show that apoNCS in its partially unfolded MG-like state resumes its native state after binding with NCS-Chr (Fig. 7). In our opinion, it may not be far-fetched to assume that in cellular environment, NCS-Chr binds apoNCS and effectively converts it into a functional protein for its own protection.
Recently, we have demonstrated apoNCS as a superior drug carrier, as its conformation is stable at wide pH range between 4–10  and is highly resistant against organic solvents and chemical denaturants . Here we further confirmed the superiority of apoNCS, as it exhibits high capability in binding and protecting the labile enediyne chromophore even under harsh acidic environment, where apoNCS conformation becomes intrinsically unstable and disrupted. How is NCS-Chr protected by the apoNCS unfolding intermediate is an interesting question. Based on the structural information obtained from NMR results (Fig. 6), residues T6, S10, S11, S14, and G16 at N-terminus, N60 and D58 at the loop region between the β-strand V (residues 53–57) and VI (residues 62–66), and S98, G104, G107, N113, and S111 at C-terminus are highly perturbed in the MG-like state of apoNCS. On the other hand, majority of the residues involved in the secondary structural interactions do not show appreciable chemical shift changes or broadening. Residues at the bottom of the chromophore binding cleft such as V34, G35, Q36, L45, G96, V95, and V108 show only small perturbation. In addition, the chemical shift of F52 and the disulfide bond C37-C47, both locate right below the nine-membered enediyne ring, are not significantly affected (Δδ of C47 is 0.094537 ppm, smaller than the 0.1 ppm threshold). It appears that many residues involved in the chromophore binding are not highly perturbed in the transition to the MG-like state. This probably accounts, at least in part, for the retaining ability of the MG-like state of apoNCS in binding and protecting the labile enediyne chromophore.
Besides being potent carrier of the natural ligand enediyne chromophore, apoNCS has also been demonstrated as a carrier of small synthetic molecules like EtBr , naphthoate ester derivatives [30, 39, 40] and flavone-based ligand . Interestingly, apoNCS is useful in improving the stability of potent DNA alkylating agents, nitrogen mustards . Furthermore, in vitro 'evolution' studies revealed that apoNCS could be engineered into a common drug delivery vehicle . Drug packaging for drug delivery systems has drawn extensive interests lately in the field of medicinal chemistry. Our study brings insight into the conserving mechanism of naturally occurring NCS and consequently merits apoNCS as a naturally built superior drug carrier for rational drug design strategies.
We identified and characterized the stable MG-like state accumulated in the equilibrium early unfolding pathway of apoNCS in the presence of 1.2 M GdnHCl under acidic (pH 3) and aqueous conditions. The apoNCS intermediate retains about 80% and 40% of the secondary and tertiary structure, respectively. With the impaired binding cleft, the MG-like state of apoNCS still exhibits full capability of protecting the labile enediyne chromophore. The results demonstrate apoNCS as the natural built-in superior drug carrier. Further CD analyses showed that NCS-Chr not only binds to the MG-like state of apoNCS but also converts the partially unfolded protein to its functional native state of holoNCS for self protection.
fast protein liquid chromatography
high pressure liquid chromatography
heteronuclear single quantum coherence.
This work was supported by a Laboratory Grant (to D.-H. Chin) (NHRI-EX90-8807BL) from National Health Research Institutes, and Individual Grants (to D.-H. Chin) (93-2320-B-005-012, 93-2113-M-005-011) from National Science Council, Taiwan, Republic of China. The work was also supported in part by National Chung Hsing University under the ATU plan funded by the Ministry of Education, Taiwan, Republic of China (to D.-H. Chin). C. Yu was supported by the National Science Council, Taiwan, Republic of China, grants (NSC94-2113-M-007-036, NSC94-2320-B-007-005). TKSK was supported by research grants from National Institutes of Health Grants (NIH NCRR COBRE Grant 1 P20 RR15569), Department of Energy (DE-FG02-01ER1516) and Arkansas Bioscience Institute, Fayetteville, Arkansas, USA. NMR experiments were carried out at the Regional Instrument Center, Department of Chemistry, National Tsing Hua University, Hsinchu, funded by National Science Council, Taiwan, Republic of China. We thank Mr. Ichiro Toishi, Kayaku Co., Ltd., for the supply of NCS powder. We thank Dr. P. Vijaya Palani for proofreading the draft and Dr. Parameswaran Hariharan for examining binding data.
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