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J. Biol. Chem., Vol. 281, Issue 23, 16025-16033, June 9, 2006

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A New Model for Ligand Release

ROLE OF SIDE CHAIN IN GATING THE ENEDIYNE ANTIBIOTIC^*

Parameswaran Hariharan, Wenchuan Liang^¶, Shan-Ho Chou, and Der-Hang Chin¹

From the Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, the Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, and the ^¶Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, California 94305

Received for publication, January 27, 2006 , and in revised form, March 20, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on ¹⁵N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe⁷⁸ mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The search for stable and nontoxic carrier-based drug delivery systems is a new trend in the recently emerging field of ligand-targeted therapeutics (1). The protein-mediated ligand-selective and high affinity carriers offer a promising strategy in drug targeting and controlled delivery of small therapeutic chemical agents (2). In light of the rapid advancements, understanding the naturally ingenious ways by which the bioactive ligands are released and controlled has been the subject of substantial interest, especially among biological chemists. The mechanism of noncovalent ligand release has been studied for many carrier proteins. Lipoprotein receptor family, -tocopherol transfer protein, retinol-binding proteins, pheromone-binding proteins, etc., are a few examples. Most models of ligand release from carrier protein complexes are hitherto found to be concomitant or subsequent to significant conformational changes in the protein structure (see Refs. 3–5 for a few examples; see Refs. 6–8 for reviews and data base).

The enediyne family of antitumor antibiotics (9, 10) includes some of the most potent antitumor agents and appears to be the only well studied category of the natural antibiotic chromoproteins. Neocarzinostatin (NCS)² (secreted by Streptomyces carzinostaticus), being the first member of this family (11), has intrigued scientists by virtue of its novel architecture, unusually high DNA cleavage activity, and antitumor effect (12). The protein component of NCS, aponeocarzinostatin (apoNCS), is biologically inactive but plays important roles in protecting and transferring the labile chromophore (NCS-C) to the target DNA (13). It is interesting to note that apoNCS does not bind to DNA (14), and its biological activity relies on the release of the tightly bound NCS-C (K_d 0.1 nM) (15) before NCS interacts with DNA. The fundamental mechanism underlying such a process displays a fascinating partnership between the biologically active chromophore and the apoprotein-based drug delivery system.

apoNCS is an all- protein with 113 amino acid residues and two disulfide bonds. It possesses a deep cavity for noncovalent binding of its chromophore (16–18). The crystal structures of apoNCS and holoNCS are reported to be almost superimposable (18). The striking similarity between the structures of apoNCS and holoNCS imply that distinct structural changes might not be necessary for the binding or release of NCS-C. We also found that chromophore release of holoNCS can precede major secondary and/or tertiary structural changes in the protein (19). This leads to a logical speculation of whether and which, if any, side chains can control the release via subtle fluctuations in protein conformation.

We anticipate that the possible candidates responsible for gating the chromophore release should satisfy two criteria. First, they must reside on the surface rather than the interior of the protein. Second, they are likely to be located in the opening of the binding cleft rather than being distributed in other parts of the surface. Physiological release of a noncovalently bound ligand from a protein complex is often triggered by a specific contact with some trans-acting agents in a cell. Such contact, if present, is more likely to occur on the solvent-exposed surface than the interior. Next, when backbone conformation remains unchanged, association or dissociation of a ligand must be through the opening of the binding cleft. Fig. 1 illustrates an aqueous model of holoNCS (20) adapted from the crystal structure (pdb.1nco.ent) (see supplementary Fig. S1 on line for the stereo view of holoNCS complex). The NCS-C is clamped in between two -hairpin loops, designated as Loop 1 (between strands 7 and 8) and Loop 2 (between strands 9 and 10). NMR studies on dynamics and relaxation of NCS reveal internal motion and localized flexibility in the loop regions around the binding cleft (21, 22). We speculate that these loop residues (residues 75–80 in Loop 1 and residues 98–102 in Loop 2) may play a pivotal role and thus set up mutagenesis studies to explore the effect of these side chains on chromophore release.

View larger version (59K): [in this window] [in a new window]   FIGURE 1. The view of an aqueous model of holoNCS complex at neutral pH (adapted (20) from pdb.1nco.ent file). The NCS-C is shown by ball and stick in red, and the protein backbone is in gray and cyan. The two loops that form the opening of the binding cleft are shown in yellow and are referred to as Loops 1 and 2. All side chains on both loops are shown in blue.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extraction of NCS-C—The NCS powder was from Kayaku Co., Ltd. (Itabashi-Ku, Tokyo, Japan). Repeated methanolic extraction of NCS-C was carried out from lyophilized NCS stock (0.5 mM) in 5 mM sodium citrate, pH 4.0, following the described method (26). The integrity and concentration of NCS-C were checked by HPLC analysis and the increased A₃₄₀ (, 10,800) after titrating with excess apoNCS.

Reconstitution of holoNCS—The reconstitution of holoNCSs was performed in ice by adding an equimolar (1:1) amount of the methanol-extracted NCS-C into a solution of recombinant protein in 5 mM ammonium acetate, pH 4.0. The mixture was incubated at 0 °C for 5–10 min to ensure complete reconstitution. The holoNCSs were freshly made before experiments, and the methanol amount in the reaction mixture was adjusted to below 5%. Care was taken to avoid degradation of the labile chromophore, and the integrity of the NCS-C after reconstitution was examined by HPLC analysis.

pCAL-n-EK-apoNCS Construct—The genomic DNA template was isolated from vegetative cultures of S. carzinostaticus (ATCC 15944). The sequence of the ncsA gene (GenBank^TM, National Institutes of Health genetic sequence data base (gi|420324|) (27), which encodes the 147-amino acid pre-apoNCS with a signal peptide at the N terminus, was used for primer design. The native gene of the 113-amino acid apoNCS was amplified by PCR with the following primers: forward, 5'-GACGACGACAAGGCGGCGCCGACGGCTACGGT-3'; and reverse, 5'-GGAACAAGACCCGTCGGAGCGGATCCTCCGATCA-3'. The 12-nucleotide upstream and 13-nucleotide downstream flanking sequences were included for ligation-independent cloning in T7/lac promoter-based Escherichia coli expression vector, pCAL-n-EK (Stratagene, La Jolla, CA). The vector (50 ng), after it is digested with Eam11041 and gel-purified, was incubated at 72 °C for 10 min in a solution containing 1 mM dTTP and Pfu DNA polymerase (ligation-independent cloning kit; Stratagene, La Jolla, CA) to produce 12- and 13-nucleotide ligation-independent cloning single-stranded overhangs. Similarly, 175 ng of the gel-purified PCR amplicons was treated with Pfu DNA polymerase in the presence of 1 mM dATP. The vector and insert were annealed at room temperature for 1 h of incubation, and the product was transformed into E. coli DH5. The apoNCS gene was fused downstream to the sequence encoding the N-terminal calmodulin affinity peptide (CBP) tag that was combined with the EK cleavage site (28, 29). The inclusion of the EK site allowed complete removal of the CBP tag and generated apoNCS with the native N terminus (i.e. Ala¹ in apoNCS). The DNA sequencing of PCR amplicons and pCAL-n-EK-apoNCS construct was performed twice on automated ABI prism sequencer model 310 (Applied Biosystems, CA) to verify the correctness of the sequence.

Site-specific Mutant Constructs of apoNCS—A PCR-based quick change mutagenesis method was applied to obtain site-specific mutant constructs of apoNCS. The codon GCG and CUG were used for the replacement to Ala and Leu, respectively, according to the codon usage of E. coli (30). Followed by PCR, DpnI was used to deplete the unmutagenized DNA. The mutant construct was transformed into E. coli DH5, and the constructed plasmids were sequenced at least twice until the correct sequence was repeatedly confirmed.

Expression and Purification of Recombinant apoNCS Proteins—The pCAL-n-EK expression construct of native or mutated apoNCSs was transformed into E. coli BL21 Codon Plus (Stratagene, La Jolla, CA). The expression of CBP-apoNCS fusion protein was induced by 0.2 mM isopropyl -D-thiogalactopyranoside at 30 °C for 5 h. After ultrasonication of the isopropyl -D-thiogalactopyranoside-induced E. coli cells, the CBP-apoNCS fusion protein was found in soluble fraction of the cell extract. The CBP-apoNCS fusion protein was purified using calmodulin affinity resin (Stratagene, La Jolla, CA) (Stratagene, La Jolla, CA) (28, 29). The protein was subsequently desalted and concentrated through Amicon or Centricon cellulose membrane (MWCO: 3000) (Millipore, Bedford, MA). The quantity of the recovered CBP-apoNCS fusion protein was evaluated by UV spectrophotometry. The extinction coefficient of the fusion protein at 278 nm, ₂₇₈ = 21,400 M^–1, was estimated from the reported value for apoNCS, ₂₇₈ = 14,400 M^–1 (31, 32), and the calculated value for the CBP tag protein, ₂₇₈ = 7000 M^–1 (ProtParam) (33). For each mg of the CBP-apoNCS fusion protein, 1 unit of EK (Invitrogen, CA, or GenScript, Piscataway, NJ) was added, and the solution was incubated at room temperature for several days. When the enzymatic cleaving of the CBP tag was more than 90% complete (checked by SDS-PAGE), the mixture was separated by DEAE-Sepharose (Fast Flow) resin (Amersham Biosciences AB, Uppsala, Sweden) with a linear gradient of 0–400 mM NaCl. The final protein product was collected after dialysis against water or filtration through Amicon or Centricon cellulose membrane (MWCO: 3000). The purified protein was analyzed by SDS-PAGE and HPLC. The averaged purity was greater than 95%. The authenticity of each protein was verified by mass spectrometry and disulfide linkage test, as described below. The final yield of recombinant apoNCS and mutants was at a range of 0.25–4 mg/liter of LB culture. Aggregation was frequently observed during purification of wild type (WT) recombinant apoNCS and mutants. This caused low yield from some mutants. For instance, the average yield of D79A was only 0.25 mg/liter of LB culture. Among all mutants purified for the study, F78L showed the highest yield (4 mg/liter of LB culture).

Expression and Purification of ¹⁵N-Labeled Proteins—Uniform ¹⁵N labeling was achieved by growing the host bacteria, E. coli BL21 Codon Plus, with vitamin B₁ in M9 minimal medium containing 0.25 g/liter of ¹⁵NH₄Cl (Cambridge Isotope Laboratories, Inc., MA) as the sole nitrogen source. The purification of the labeled apoNCS and mutants was carried out following the same procedure as described above. The yield was about half of that of the corresponding unlabeled protein. Changing ammonium chloride from 0.50 to 0.125 g/liter of medium did not significantly reduce or improve the protein yield.

Mass Spectrometry—All of the mass spectrometries were done on a Finnigan LCQ mass spectrometry detector (Thermo Electron, San Jose, CA) equipped with atmospheric pressure ionization source using electrospray ionization (+)-charge mode. The protein samples were diluted in 30% acetonitrile containing 0.1% trifluoroacetic acid for analysis.

NMR Spectroscopy—One-dimensional ¹H NMR spectra of unlabeled WT apoNCS and mutants were recorded at room temperature on a Varian 600-MHz NMR spectrometer (Palo Alto, CA). The two-dimensional ¹⁵N-¹H heteronuclear single quantum coherence (HSQC) NMR spectra were recorded on a Bruker DMX 600-MHz spectrometer (Rheinstetten, Germany) at 25 °C. The NMR samples were prepared by dissolving lyophilized protein in 20 mM sodium phosphate, pH 7.0, in 10% D₂O, 90% H₂O. The cross-peaks for WT apoNCS were identified based on the reported assignments (25). The cross-peak of Tyr³², which is in a tyrosine corner-like motif and is considered to contribute stability of apoNCS (34), was used here as an internal reference.

CD Spectroscopy—All of the CD measurements were carried out on a Jasco J-715 spectropolarimeter (Tokyo, Japan) equipped with a circulating water bath (Neslab, model RTE-140) (Portsmouth, NH). For apoNCS and its mutants, a 200-µl protein solution in 20 mM sodium phosphate, pH 7.0, with a concentration level of 10–15 µM for far UV CD and 50 µM for near UV CD, was used for measurement. The spectrum was recorded at 25 °C using a temperature controlled water-jacketed cell with 0.1-cm-path length at a scan speed of 20 nm/min. For reconstituted WT and mutated holoNCSs, a 10 µM sample in 20 mM ammonium acetate, pH 4.0, was used for measurement. Because of the instability of holoNCSs, the scan speed was increased to 100 nm/min to reduce time. All of the spectra were accumulated minimum five times and are corrected for the respective buffer blanks. The data are expressed as the mean residue ellipticity for both apoNCS and holoNCS (19).

Fluorescence Spectroscopy—The fluorescent changes from a holoNCS sample in kinetic release study were monitored by a SLM Aminco Bowman Series II luminescence spectrometer (SLM Aminco Bowman, Urbana, IL). The sample temperature was controlled at 25 °C by a thermostatic cell holder equipped with a refrigerated thermostatic bath circulator. The sample of volume 150 µl in a 3-mm square cell was used. A single point acquisition of emission data at 440 nm was collected once per 30 s by excitation at 340 nm. To avoid the degradation of holoNCSs by excessive exposure to the light source, the light shutter was controlled by modification of time scan mode using an in-house written macro language commands so that it can be closed in between data collections. In a 100-min monitoring, exposure of NCS to light was minimized to 2.33 min. A rapid kinetics setup was used for fast releasing mutant F78L, where the shutter control was suspended for the initial 3 min to collect data once per 5 s. After that, the shutter control was enabled as described.

Disulfide Linkage Test—A facile disulfide evaluation procedure was developed for checking the integrality of the produced recombinant apoNCS and its mutants. To 100 µl of solution of 10 µM apoNCS protein in 0.1 M sodium phosphate, pH 8.0, guanidine hydrochloride was added to a final concentration of 4 M. After mixing, 80-fold molar excess of iodoacetamide over apoNCS (20-fold excess/Cys residue) was added at 37 °C for 2 h. Under this treatment, alkylation can occur on any un-oxidized Cys residues in apoNCS. The protein sample was then desalted and analyzed by mass spectrometry.

Thermal-induced Denaturation—The thermal-induced denaturationof50 µM of WT apoNCS and mutants, in 10 mM sodium phosphate buffer, pH 7, was monitored by CD spectroscopy at 224 nm from 25–91 °C with 3 °C increments. The temperature was controlled by a microprocessor and a temperature sensor. The equilibrium time was 15 min, and the ellipticity was recorded for 30 s at each temperature setting.

Kinetic Study on the Release of NCS-C—The rate of NCS-C release from natural, reconstituted WT or mutated holoNCSs was determined using the method previously published (19). The reconstituted holoNCSs (10 µM) were prepared in 100 mM Tris-HCl, pH 7.0, and the release of NCS-C was monitored by fluorescence spectroscopy at 25 °C after the addition of 5 mM GSH. The final reading of the emission was obtained after the sample was treated with 80% isopropanol (to complete the release of NCS-C). The release kinetics was also independently examined by HPLC analyses. The samples (0.5 nmol), prepared as above, were drawn at different time points and were analyzed by reverse phase HPLC. The quantity of the intact NCS-C and GSH-induced adduct, which represent population of the protein-bound and released species, respectively, were estimated using the method described (35).

Kinetic Release Data Processing Information—The rate of release is expressed as

(Eq. 1)

where k_obs and n are the observed rate constant and reaction order for the rate of disappearance of holoNCS, respectively, and k₁ is the first order rate constant after approximation. However, the release rate decreased gradually with time when ratio of apoNCS to NCS-C increased with progressing lose of the intact NCS-C. For consistency, all of the rate constants were calculated from the initial 10% release that fit into a first order linear plot (the averaged r = 0.97 analyzed by the Kaleidagraph program (Synergy Software, Reading, PA)).

holoNCS-induced DNA Damage—The DNA cleavage ability of each chromoprotein complex was assessed by the proportion of conversion from form I DNA (supercoiled) to form II (relaxed; caused by single strand breaks) and form III (linear; caused by double-stranded breaks). The supercoiled pBR322 DNA was isolated from E. coli using Mini-Prep plasmid isolation kit (Qiagen Inc.). A 16-µl DNA drug reaction mixture was prepared by mixing 5 µM (final concentration) of freshly reconstituted holoNCS, 5 mM GSH, 100 mM Tris-HCl, pH 7.0, and 40 ng/µlof pBR322 DNA. The mixture was incubated at 16 °C for 15 min and was immediately loaded onto a 1% w/v agarose gel for analysis.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. The structural characterizations of WT and mutants of NCS. A, the far UV and near UV CD (inset) spectra of apoNCSs reveal identical pattern. B, the CD spectra of reconstituted holoNCSs suggest similar NCS-C binding capabilities. In A and B, the spectra shown are obtained from recombinant WT (), F76A (— -), L77A (- - -), F78A (— —), F78L (- - -), D79A (·- ·-), S98A (·······), and D99A (·· - –). C, the thermal denaturation, monitored by ellipticity changes at 224 nm, indicates compatible stability of WT and mutated apoNCSs. The profiles shown were obtained from: recombinant WT (•), F76A (), L77A (), F78A (), F78L (), D79A (), S98A (), and D99A ().

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning and Characterization of Recombinant apoNCS Proteins—After a long attempt, we successfully isolated the native gene of the 113-amino acid apoNCS from S. carzinostaticus. The use of pCAL-n-EK allowed expression of a fusion protein from which apoNCS could be retrieved by a proteolytic removal of its tag by EK. The recombinant apoNCS thus obtained possessed the native termini without any extraneous residues. After DEAE-Sepharose ion exchange chromatography, the purified recombinant apoNCS and mutants showed a single band corresponding to the band of the natural apoNCS in SDS-PAGE. A single peak was observed by HPLC analysis, indicating the homogeneity of the purified proteins. The mono isotopic mass number of the recombinant WT apoNCS (11085 ± 1), as determined by electrospray ionization-mass spectrometry measurement, matches with the theoretical value (11085.2). The mono isotopic mass numbers of all mutants and ¹⁵N-labeled proteins are also in good agreement with their theoretical molecular weights.

Disulfide Assay—To avoid any possible artifacts, it was crucial to examine whether all disulfide linkages were properly formed in the produced proteins. This assay was specifically developed for apoNCS, and the sensitivity and validity have been evaluated and confirmed.³ As evidenced by mass spectrometry measurement, no alkylated species was observed for all purified proteins obtained in the current investigation. The results demonstrate the absence of free–SH in cysteine residues and indicate that the WT and all mutated apoNCSs contain two disulfides as the natural one does.

CD Spectra of WT and Mutated apoNCS—The structural characterization of WT and mutant apoNCSs was carried out by CD. The far UV CD spectrum of the recombinant WT apoNCS (Fig. 2A) revealed a predominant -sheet structure. The profile is consistent with that reported for the native apoNCS (19, 23, 36, 37). The far UV CD spectra of F78A, F78L, D79A, and S98A mutants also closely resembled that of WT apoNCS. Only a slight deviation was observed in the spectra of F76A and D99A mutants. Overall, the characteristic features of the far UV CD of WT apoNCS were retained in all of the mutants, suggesting WT-like secondary structures existing in all the mutants.

Similarly, the near UV CD spectrum (Fig. 2A, inset) of recombinant WT apoNCS exhibited the same broad negative signal around 271 nm as observed for native apoNCS. All of the mutants exhibited a similar broad negative band with similar intensity in the mean residue ellipticity. The results reveal that the aromatic side chains of Tyr³², Trp³⁹, and Trp⁸³ experience the same steady and compact tertiary environment as seen in the native apoNCS.

Thermal Denaturation Experiments—To compare the conformational stability with that of natural apoNCS, thermal denaturation of recombinant WT and all of the mutated apoNCSs was studied by CD spectroscopy. The T_m of the recombinant WT apoNCS is shown as 64.0 °C, which is consistent with the T_m (63.5 °C) of the natural apoNCS measured by CD (37) but is slightly lower than the T_m (68 °C) measured by differential scanning calorimetry (38). The denaturation curves (Fig. 2C) show that both side limbs (25–52 and 73–91 °C) and the rapid unfolding phase (52–73 °C) are nearly the same for all mutants. The measured T_m values are 63.5, 58.0, 65.5, 65.7, 60.4, 64.5, and 63.3 °C for F76A, L77A, F78A, F78L, D79A, S98A, and D99A mutants, respectively. The observed T_m values for L77A and D79A are slightly smaller (6 and 4 °C) than that of WT apoNCS. This is not surprising, considering the well known fact that charged residues at surface can stabilize proteins (39). Likewise, replacing the large hydrophobic residues with small ones can also reduce stability (39, 40).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. The role of Phe78 side chain in gating the release of NCS-C. A, the kinetic release of NCS-C from reconstituted holoNCSs reveals the sensitive position of Phe78. The release, after adding excess GSH, was monitored at pH 7 and 25 °C through fluorescence changes at 440 nm (excitation at 340 nm), arising from the irreversible inactivation of the freed NCS-C by GSH. The profiles shown were obtained from holoNCSs made of WT (•), F76A (), L77A (), F78A (), F78L (), D79A (), S98A (), and D99A (). B, HPLC-based quantification analyses on the remaining protein-bound NCS-C show the same trend in release rate. The time course quantification profiles shown were obtained from holoNCSs made of WT (•), F78A (), and F78L (). All of the other mutants exhibit similar profiles as WT (data not shown). C, in vitro DNA cleavage mediated by reconstituted WT and mutated holoNCSs demonstrates that Phe78 mutants enhance biological activity of NCS. The drug-induced cleavage of the pBR322 plasmid is analyzed by 1% agarose gel electrophoresis. Form I, II, and III denote supercoiled, relaxed circular, and linear forms of DNA, respectively. The arrow indicates the accumulation of the cleaved DNA fragments by F78L mutant.

Kinetic Release Monitored by Fluorescence—GSH failed to interact with the protein-bound NCS-C (20) but could quickly inactivate freed NCS-C by forming an adduct that generates 60-fold stronger fluorescence than NCS-C does (35). This enabled us to monitor the release kinetics on the basis of changes in fluorescence. In the presence of excess GSH (5 mM), a sample of 10 µM reconstituted WT holoNCS, at 25 °C and pH 7, releases 22% of NCS-C in a period of 100 min. Under the same condition, a similar release rate was obtained from the natural holoNCS, confirming the validity of our method of reconstitution of holoNCS. Fig. 3A shows the release profiles of reconstituted WT and mutated holoNCSs. All of the mutants, except those at Phe⁷⁸, have equal or even slightly suppressed release rate (for example, F76A and S98A) compared with that of WT. A striking increase in the chromophore release was observed only in Phe⁷⁸ mutants. The mutated holoNCS reconstituted from F78L released 90% NCS-C within 15 min under the same condition and hence exhibited the fastest rate among all.

The stability of NCS is known to be influenced by its concentration and molar ratio of NCS-C to apoNCS (14). Because NCS-C is labile only when released, this fact implies that the release rate should also rely on these factors. Because the released NCS-C is continuously inactivated by excess GSH in solution, accumulation of free apoNCS molecules causes molar ratio of apoNCS to NCS-C to increase and thus gradually slows down the release rate. For the purpose of comparison, all of the rate constants herein were obtained from 10 µM of freshly reconstituted chromoproteins and were calculated from the initial 10% NCS-C release that fit into a first order linear plot (Table 1). Except for Phe⁷⁸ mutants, all of the other mutants showed release rates comparable with that of WT holoNCS (k = 0.19 h^–1), with variations in rate no larger than 20%. Some mutants, such as S98A, even showed a slightly suppressed release rate than WT. However, an amazingly large increase of nearly 7- and 90-fold in release rate was constantly obtained in mutants F78A and F78L, respectively.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Evaluation of the binding structural integrity at the residue level on the fast releasing mutants of NCS. Two-dimensional 15N-1H HSQC NMR spectra of WT and Phe78 apoNCS mutants at 25 °C and pH 7 are compared with identical axis scales. The cross-peaks were assigned based on the reported chemical shifts (25). The cross-lines in the spectrum of WT, F78A, and F78L apoNCS show that residues involved in NCS-C binding are barely perturbed by mutations.

Two-dimensional ¹⁵N-¹H HSQC NMR Experiments—Two-dimensional ¹⁵N-¹H HSQC NMR spectroscopy enables one to closely inspect the changes in binding pocket structure, in particular in those mutants that show enhanced chromophore release. We therefore carried out NMR experiments for WT and mutant apoNCSs under conditions similar to that of release experiments. The ¹⁵N and ¹H chemical shifts of apoNCS at pH 7 showed good agreement with those at pH 5 (25), as predicted by the fact that apoNCS is stable in a wide pH range of 4–10 (37). Close comparisons among the ¹⁵N-¹H HSQC spectra obtained from WT, F78A, and F78L apoNCS are shown in Fig. 4. The results reveal that most cross-peaks remain dispersed and unperturbed by mutations along the entire region. The chemical shift of residues at both terminals, which are, in general, more dynamic than other portions, also appears to be unchanged upon mutation. The Thr⁴ residue at the N terminus and Asn¹¹³ at the C terminus shown in Fig. 4 are such examples. A number of residues, including Gly³⁵, Trp³⁹, Leu⁴⁵, Pro⁴⁹, Phe⁵², Phe⁷⁶, Leu⁷⁷, Gly⁸⁰, Gln⁹⁴, Val⁹⁵, Gly⁹⁶, Ser⁹⁸, Ala¹⁰¹, and Gly¹⁰² can form van der Waals' contact or H-bond with NCS-C (18, 42). Among those, the residues that have close contacts with naphthoate group of NCS-C, namely, Phe⁵², Ser⁹⁸, Gly⁹⁶, Gln⁹⁴, and Trp³⁹, are particularly important for binding (43). Fig. 4 also shows that the resonances from these binding residues remain mostly unchanged upon mutation at Phe⁷⁸. There are a few perturbations observed in the residues neighboring the point mutation in both mutants. A small shift at the cross-peak of Gly⁸⁰ in the F78A mutant is one example. Such changes are common phenomena (40) and are often not significant in changing the side chain packing in a mutant (44). The results of HSQC NMR experiments revealed that the overall binding backbone conformation of apoNCS is unchanged, despite mutation at Phe⁷⁸. This excludes the possibility that the fast releasing caused by the mutation at the side chain of Phe⁷⁸ results from the backbone conformational changes in the binding cleft.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. A new model of drug release illustrated by the enediyne containing antibiotic chromoprotein NCS. The schematic surface view (pdb.1nco.ent) of holoNCS (left) and apoNCS (right) shows the strategic bridging position of the benzene ring of Phe78 (shown in blue) in gating the release of NCS-C (shown in yellow). The flipping motion between the "open" and "closed" conformation of Phe78 could be triggered by a release stimulant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When a protein-bound ligand is freed without a major preceding change in protein conformation, the ligand must escape through an existing "opening." The residues around the opening at the protein surface therefore play a major role in gating the release. In this study, we focus on mutation in NCS around the opening of the binding pocket, constituted mainly by two loops (Gly⁷⁵-Phe⁷⁶-Leu⁷⁷-Phe⁷⁸-Asp-⁷⁹-Gly⁸⁰ in Loop 1 and Ser⁹⁸-Asp⁹⁹-Ala¹⁰⁰-Ala¹⁰¹-Gly¹⁰² in Loop 2) (Fig. 1). The small residues, such as Gly⁸⁰, Ala¹⁰⁰, Ala¹⁰¹, and Gly¹⁰², were excluded from the alanine-scanning mutagenesis employed in this study. Because all of the mutants except Phe⁷⁸ show no significant effect, the inclusion of leucine replacement of Phe⁷⁸ brings one more factor for our current investigation.

Although employing an E. coli-derived recombinant protein as a substitute for the natural NCS produced by S. carzinostaticus, we avoided bringing in any extraneous residues at the termini to dismiss any possible obscurity in protein conformation. Disulfide assay demonstrated that disulfide linkage is properly formed in all of the produced proteins. Structural comparison of WT and mutated apoNCSs indicated that they are similar in secondary and tertiary structures, as evidenced by the results of CD spectroscopy (Fig. 2A). Moreover, the closely matching T_m values of the mutants show their almost equal thermal stability (Fig. 2C). To examine the possible changes in the specific interaction between NCS-C and apoNCS, we performed two-dimensional NMR using ¹⁵N-labeled proteins, to closely examine the perturbations caused by mutations in the residues that account for the affinity of the chromophore. The results show that most resonances of Phe⁷⁸ mutants overlap with those of WT (Fig. 4), suggesting that the overall architecture of the protein and the interior binding pocket were barely affected by Phe⁷⁸ mutations. This excludes the possibility that the fast release from Phe⁷⁸ mutated proteins resulted from perturbations in the binding interaction. The ability of all mutants to bind NCS-C is further supported by the results of CD studies (Fig. 2B). All of the mutants show a similar chromophore-binding pattern that is close to that of WT. The results indicate that NCS-C binding can efficiently occur, despite these mutations at the side chains around the opening of the binding pocket.

The release of NCS-C in vivo and its action on host DNA disturbed the equilibrium between apoNCS and holoNCS by constant dissociation and inactivation of NCS-C. The time course of this inactivation determined the biological availability of the active chromophore (14). To mimic this biological action, we measured the time course release in the presence of excess GSH, which can efficiently inactivate the released, but not the protein-bound, NCS-C. The fluorescence-based kinetic experiments (Fig. 3A and Table 1) clearly demonstrate that mutations at Phe⁷⁸ caused large increase in release rate, whereas other mutations around the opening of the binding cleft caused no significant effect. Quite interestingly, the mutations of the residues, Phe⁷⁶, Leu⁷⁷, and Ser⁹⁸ that are predicted to be involved in NCS-C binding through hydrogen bond or van der Waals' contact (18, 42) were found to cause only minimal changes in release rates. The time course HPLC analyses on the amount of the remaining protein-bound NCS-C revealed the same trend in release rate (Fig. 3B). Consistent with the kinetic results, the data of DNA cleavage experiments correlate well with the enhanced cleavage observed in fast releasing Phe⁷⁸ mutants (Fig. 3C). All of the results consistently pinpoint the significant role of the Phe⁷⁸ side chain in gating the chromophore release, when major backbone conformation of the protein remains unchanged. Currently we do not have a proper explanation for the difference in releasing property among Phe⁷⁸ mutants. This will rely on continuous efforts involving more mutagenesis studies at the Phe⁷⁸ position.

The importance of Phe⁷⁸ in NCS-C binding has been previously suggested (46). NMR studies indicate that the aromatic ring of Phe⁷⁸ is in van der Waals' contact with one triple bond of NCS-C (42). In crystal structures, the only noticeable difference between apoNCS and holoNCS is the position of the Phe⁷⁸ ring (18). In holoNCS, the aromatic side chain of Phe⁷⁸, which engages in a -interaction with the enediyne ring of NCS-C, is bent toward the binding cleft. In apoNCS this side chain is opened and turned away from the binding cleft to have a solvent-exposed position. On the other hand, nuclear Overhauser effect data and model studies indicate that Phe⁷⁸ takes multiple conformations in solution (47). Relaxation studies suggest that residues 72–87 experience small amplitude motions on a picosecond fast time scale (21). Although Phe⁷⁸ appears to be quite dynamic from these NMR data, a newer three-dimensional NMR study (48) suggests that there is no significant internal motion for the loop 75–84, and the side chain of Phe⁷⁸ has a rigid conformation. Despite some contradictions, Phe⁷⁸ has been consistently considered to be one of the critical residues responsible for NCS-C stabilization.

The present findings provide direct evidence that mutations at Phe⁷⁸ have a drastic effect on accelerating NCS-C release at large magnitude. The results lead us to propose a releasing model involving the side chain of Phe⁷⁸. Fig. 5 shows a surface view of holoNCS in a similar orientation as shown in Fig. 1. The view illustrates Phe⁷⁸ as a bridge lying between Loops 1 and 2, blocked in the middle of the opening of NCS-C binding cleft. This implies the strategic and sensitive position of Phe⁷⁸ in gating the chromophore release. When the conformation of Phe⁷⁸ side chain is fluctuated by breathing-like dynamic motion or perturbed by any as yet unknown stimulant in a cellular environment, the aromatic ring can rotate or change orientations. Under these circumstances the bridged bar made by Phe⁷⁸ side chain could be transiently opened. The distance of the cleft between Loops 1 and 2 (10.38 Å, between C of Phe⁷⁸ and C_methyl of Ala¹⁰⁰) is sufficiently large to allow NCS-C to escape through the wide width of the opening. This model provides a release mechanism in which the bound NCS-C can be released without changing any major backbone conformation of the protein. It should be noted that NCS-C is not sustained solely by Phe⁷⁸. Residues at the bottom of the binding cleft also involve in the NCS-C binding.

Despite its novelty, the present model does not rule out the alternative major conformational change-dependent release of chromophore from NCS. Major protein conformational changes can facilitate NCS-C release, although they are not required. Whether any major structural changes are involved in NCS action in vivo is not yet known. A "sliding" model is proposed recently for chromophore-releasing mechanism, which involves certain backbone conformational changes in protein (48). Lately, we find that apoNCS is highly resistant to denaturants (49). We have nearly completed an identification of a release triggering mechanism under the cell surface mimetic conditions demonstrating that the release of NCS-C is triggered by means of major conformational change independent fashion.⁴ These observations might favor the lack of requirement of a major conformational change for the release of NCS-C.

Antibiotic chromoproteins are naturally occurring special teams with interesting features. Such combination involves a labile cytotoxic chromophore protected by a carrier protein, which differs from that of the usual prosthetic chromoproteins such as cytochromes. An antibiotic chromoprotein needs to serve a paradoxical situation; although it exhibits a similar high affinity for its ligand as heme proteins, it must also "release" the toxin in a correct spatiotemporal manner to exert its antibiotic function. If the present model of drug release signifies the in vivo events, it highlights the ingenuity of nature in accomplishing mutually exclusive complex functions of a macromolecule by a simple manipulation, achieving two contradictive roles of a carrier, i.e. stabilizing the bound drug and releasing the same by operating the side chain of a critical amino acid. apoNCS and its evolution variants are able to carry a number of small molecules such as ethidium bromide (50), nitrogen mustard (51), or testosterone (52). Taking into account of the enhanced DNA cleavage by Phe⁷⁸ mutants, the present drug release model provides a promising indication for engineering pharmacological packages in a broad functional context.

	FOOTNOTES

 
^* This work was supported by Laboratory Grant NHRI-EX90-8807BL (to D.-H. C.) from the National Health Research Institutes and Individual Grants 93-2320-B-005-012 and 93-2113-M-005-011 (to D.-H. C.) from the National Science Council, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan. Tel.: 886-4-22840411, Ext. 304; Fax: 886-4-22862547; E-mail: chdhchin{at}dragon.nchu.edu.tw.

² The abbreviations used are: NCS, neocarzinostatin; holoNCS, holoneocarzinostatin; apoNCS, aponeocarzinostatin; NCS-C, NCS chromophore; WT, wild type; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum coherence; EK, enterokinase; CBP, calmodulin-binding peptide.

³ C.-J. Tseng and D.-H. Chin, unpublished data.

⁴ P. Hariharan., S.-H. Chou, and D.-H. Chin, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank the Regional Instrumentation Centers (funded by National Science Council, Republic of China) at the Department of Chemistry, National Chung Hsing University, Taiching, and National Tsing Hua University, Hsinchu, for carrying out NMR experiments. We thank the Kayaku Co., LTD., for the supply of NCS powder. We thank Dr. Joseph A. J. Raja for proofreading the manuscript. Special thanks to Prof. Robert L. Baldwin, Stanford University, for many valuable suggestions.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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