Structural Insight into the Self-Sacrifice Mechanism of Enediyne

Aug 18, 2006 - Facility at Madison, Biochemistry Department, University of ... National Cooperative Drug Discovery Group, School of Pharmacy, Universi...
0 downloads 0 Views 1013KB Size
Download PDF
ARTICLE

Structural Insight into the Self-Sacrifice Mechanism of Enediyne Resistance

Shanteri Singh†,‡, Martin H. Hager‡, Changsheng Zhang‡, Byron R. Griffith‡, Min S. Lee†,储, Klaas Hallenga§, John L. Markley,†,§ and Jon S. Thorson‡,¶,* † Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706-1544, ‡Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, Wisconsin 53705, §National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin–Madison, 433 Babcock Drive, Madison, Wisconsin 53706, and ¶University of Wisconsin National Cooperative Drug Discovery Group, School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, Wisconsin 53705, 储Present address: MinHak Pharmaceuticals, 6356 Paseo Cerro, Carlsbad, California 92009.

T

he enediyne antitumor antibiotics (Figure 1), are characterized structurally by an enediyne core unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a 9- or 10-membered ring (1–4). To date, six naturally occurring 10-membered- (Figure 1, 1–6), and five 9-membered- (Figure 1, 7–11), or “chromoprotein” enediynes, have been elucidated structurally (5–7). In general, these enediynes contain three distinct structural elements: a DNA-recognition unit (e.g., the aryltetrasaccharide of 1), which serves to deliver the metabolite to its target DNA; an activating component (e.g., the methyl trisulfide of 1), which sets the stage for cycloaromatization; and the enediyne “warhead”, which cycloaromatizes to a highly reactive diradical species (e.g., Figure 1, species II) and, in the presence of DNA, results in oxidative strand scission of the targeted sequence (8–11). In vitro and in vivo studies are consistent with the role of enediynes as DNA-damaging agents and suggest that they may even favor cleavage at certain chromosomal sites and/or tertiary structures (12, 13). Although this extraordinary reactivity invokes incredible potency (some enediynes are ⬎8000-fold more potent than adriamycin), the enediynes are similar to most cytotoxics in their general lack of specificity. However, the clinical success of enediynes has been derived via targeting with tumor-specific monoclonal antibodies (mAb) (as in the 1-based MyloTarg to treat acute myelogenous leukemia) (14–17) or through the application of polymer-assisted delivery devices (such as 1-poly[styrene-maleic acid]-conjugated 8). www.acschemicalbiology.org

A B S T R A C T The recent discovery of the first “self-sacrifice” mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products (e.g., bleomycins, mitomycins, and ninemembered chromoprotein enediynes), this is the first demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct hydrogen abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the first time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.

*Corresponding author, [email protected].

Received for review July 8, 2006 and accepted July 28, 2006. Published online August 18, 2006 10.1021/cb6002898 CCC: $33.50 © 2006 by American Chemical Society

VOL.1 NO.7 • ACS CHEMICAL BIOLOGY

451

Figure 1. Naturally occurring enediynes: 1, CLM ␥1I, (fragment III is highlighted by the bracket, and cycloaromatized products I, dihydrothiophene, and II, CLM ⑀, are also illustrated); 2, shishijimicin A; 3, namenamicin; 4, esperamicin A1; 5, dynemicin A; 6, uncialamycin; 7, C-1027; 8, neocarzinostatin; 9, kedarcidin; 10, maduropeptin; 11, N1999A2.

Recently, the locus encoding for calicheamicin (CLM) biosynthesis in Micromonospora echinospora was discovered by screening a M. echinospora genomic library for cosmids that conferred CLM resistance (6, 18, 19). Preliminary analysis of these cosmids revealed a single gene (calC) responsible for CLM resistance. Subsequent in vitro studies of CLM inactivation by CalC revealed the mechanism of inactivation to proceed via abstraction of a CalC Gly113 C␣-hydrogen by the transient enediyne diradical species, thereby quenching the reactive enediyne moiety while generating a CalC Gly113 C␣ radical (Figure 2) (19). Reminiscent of the mechanism of enediyne-based DNA scission (20), this CalC Gly113 C␣ 452

VOL.1 NO.7 • 451–460 • 2006

SINGH ET AL.

radical subsequently reacts with oxygen to ultimately provide oxidative site-specific proteolysis of CalC. This cumulative reaction, wherein the sacrifice of the CalC protein accompanies CLM inactivation, was noted as the first “self-sacrifice” mechanism of antibiotic resistance. CalC was also shown to inactivate two other members of the 10-membered enediyne family, shishijimicin and namenamicin (Figure 1, 2 and 3, respectively). In an effort to further our understanding of this distinctive CalC enediyne inactivation mechanism, we pursued the structural analyses of CalC and the CalC–CLM complex. The studies reported herein reveal CalC to be a member of the steroidogenic acute regulatory protein (StAR)www.acschemicalbiology.org

ARTICLE related transfer (START) (21, 22) domain structural superfamily and to contain a small DNA-binding region. The potential implications of these findings to the CalC selfsacrifice mechanism and possibly even the post-endocytotic intracellular transport of enediyne-based therapeutics are also discussed. RESULTS AND DISCUSSION Overview of the CalC Structure. The 15N-HQSC spectrum of fulllength CalC1–181 indicated the first 30 residues of CalC to be unstructured as predicted by the PSI-PRED protein structure prediction server (23). N-terminal truncation of CalC provided a protein (CalC27–181) that was functionally equivalent and that gave a 15N-HQSC signature that could be superimposed with that of the full- Figure 2. The competing pathways of the enediyne-induced DNA strand scission that leads to cell death length protein (Supplementary (top) and the CalC self-sacrifice enediyne resistance mechanism (bottom). Figure 1). Given the minimal influence of the 26 N-terminal residues upon activity or struc- straints obtained from TALOS (26) on the basis of backtural integrity, CalC27–181 was employed for all studies bone chemical shift values and 100 hydrogen bond constraints deduced from characteristic NOEs of the secdescribed herein and for simplicity is referred to as ondary structures (see Methods). The 20 conformers “CalC”. The CalC structure was determined at 30 °C with the lowest total energy were used for final analysis. (0.8 mM CalC, 10 mM NaPO4, pH 7.3, and 150 mM NaCl) by multidimensional heteronuclear NMR spectros- All conformers exhibited good geometry, no violations of distance restraint ⬎0.5 Å, and no dihedral angle violacopy of uniformly 15N- and 15N/13C-labeled protein tions ⬎5° (Supplementary Table 1). The final superimsamples. On the basis of gel filtration and NMR relaxposed ensemble of 20 conformers (Figure 3, panel a) ation measurements, CalC was found to exist as a monomer in dilute solution with a tendency to aggregate had root mean square deviation (rmsd) values (over residues 27–181) of 0.65 ⫾ 0.11 Å for backbone heavy above ⬃1 mM CalC. Nearly complete 1H, 15N, and 13C assignments were obtained from analysis of data from a atoms and 1.16 ⫾ 0.12 Å for all heavy atoms. standard set of NMR experiments. The global fold was The CalC secondary structural elements (Figure 3) established unambiguously from assigned chemical include three ␣-helices, one 310-helix, and seven antishifts and long-range nuclear Overhauser effects (NOEs). parallel ␤-strands. The seven ␤-strands form a concave Distance constraints were derived as described in open barrel that is closed by helix ␣1 from the Methods. The program CNS (24) with ARIA (25) was N-terminus, two capping helices ␣2 and ␣3 from the used to calculate an ensemble of 100 conformers of C-terminus, and L1 loop. This fold provides a large CalC by simulated annealing on the basis of 2518 hydrophobic cavity that spans nearly the entire length of unambiguous and 248 ambiguous distance constraints. CalC. The hydrophobic core contains aromatic side Additional constraints used in the final calculation of chains stacked on the aliphatic side chains of other hydrophobic residues. These interactions are mani100 conformers consisted of 160 dihedral angle conwww.acschemicalbiology.org

VOL.1 NO.7 • 451–460 • 2006

453

proteins (29), and human/murine lipid binding proteins (Table 1). These structural homologues are recognized as part of the START superfamily of proteins, the members of which characteristically bind hydrophobic ligands and frequently appear in multidomain proteins where they regulate the activity of associated domains (21, 22). The START superfamily encompasses a wide range of bacterial, archaeal, and eukaryotic proteins that have Figure 3. NMR solution structure of CalC. a) Backbone stereoview of the been implicated in a variety of cellular functions, includensemble of 20 conformers that represent the structure (residues 27–181). Coloring scheme: blue, ␤-strand residues (residues 32ⴚ38, 71ⴚ73, 78ⴚ81, ing lipid transport and metabolism, signal transduc89ⴚ98 with bulges at Arg95, 102ⴚ106, 123ⴚ130, and 134ⴚ141); red, ␣tion, and transcriptional regulation (21, 22, 30, 31). helical residues (residues 41ⴚ50, 149ⴚ156, and 163ⴚ176); yellow, 310-helix A unique structural feature conserved within START (residues 53ⴚ55); gray, other residues. b) Ribbon representation of the domains is a hydrophobic cavity in which subtle modifirepresentative structure of CalC (the conformer closest to the average). cations dictate ligand/substrate specificity. Apart from Numbers represent the order of ␤-strands, ␣-helices, and loops (overall order ␤1-␣1-310-␤2-␤3-␤4-␤5-␤6-␤7-␣2-␣3), and the N- and C-termini of the this, the START superfamily is characterized by notable protein are indicated. The molecular graphics program MOLMOL was used in structural variations, including the absence or distortion generating these views of the structure. of the first ␣-helix, differences in the number of strands forming the ␤-sheet, and the size of the hydrophobic fested by low-frequency chemical shifts of protons from tunnel (30). Consistent with this familial variation, CalC several methyl groups. For example, ring current effects contains two ␣-helices at the C-terminus (␣2 and ␣3), from Phe127, Phe123, and Phe53, respectively, account whereas the closest CalC structural homologues each for abnormal chemical shifts observed for the methyl possesses a single continuous C-terminal helix. As disprotons of Val137, Val139, and Ile166. cussed below, the unique C-terminal structural feature Similarity to the START Domain Superfamily. There of CalC appears to be involved in DNA binding. The CalC–CLM Complex. CLM (Figure 1 and Figure 2), are no CalC sequence homologues, and all de novo which contains a core bicyclo (7.3.1) tridecadiynene sequence-based models failed to provide any CalC moiety (enediyne warhead) appended by an aryltetstructural insights. The minimum energy structure of rasaccharide chain (6), is highly hydrophobic and is CalC was submitted to the fold recognition programs DALI (27) and VAST (28). From this analysis, the closest sparingly soluble in aqueous buffer. We used the NMR chemical shift perturbation method (32) to determine structural homologues identified displayed very low the interaction surface of the CalC protein with CLM. A sequence identity to CalC (4–13%) and include a proseries of 2D-1H–15N heteronuclear single quantum corkaryotic protein with unknown function, plant allergen relation (HSQC) spectra of [U-15N]-CalC in 15% dimethyl sulfoxide (DMSO) were recorded to follow TABLE 1. Structural homologues of CalC the effects of titration with ligand/substrate from a CLM-DMSO stock solution. Chemical shift changes PDB Z-score rmsd ( Å) % Identity Lengtha Protein upon titration identified residues that interact 1XUV 12 3.1 16 160 Unknown directly with CLM along with those affected indi1XFS 11 3.3 13 149 Unknown rectly by substrate/ligand-binding (Supplementary 1XN5 8.2 3.1 17 138 Unknown Figure 2). Upon CLM titration, chemical shift 1BV1 8.4 2.9 9 126 Plant allergen changes were observed mainly in the loop regions, 1VJH 6.7 3.4 13 102 Plant allergen indicating their involvement in the interaction with 1JSS 6.7 3.6 4 110 Lipid binding (STARD4) CLM (Supplementary Figure 2). However, although 1EM2 6 3.3 5 111 Lipid binding (STARD3) the chemical shift perturbations provided a general 1T17 5.6 4.1 9 148 Unknown indication of the CalC–CLM interaction surface, the 1ND0 4.3 3.3 10 109 Non-heme iron dioxygenase limited solubility of CLM, even in 15% DMSO, complicated more precise refinement of the CalC–CLM a Sequence length over which percentage sequence identity is calculated. complex structure. 454

VOL.1 NO.7 • 451–460 • 2006

SINGH ET AL.

www.acschemicalbiology.org

ARTICLE To obtain a more accurate and quantitative measurement of the protein–ligand interaction, a more soluble CLM fragment (Figure 1, fragment III) was employed. Titration of [U-15N]-CalC with fragment III (Figure 4) followed by NMR 2D-1H–15N HSQC, identified a set of residue perturbations similar to those previously found with CLM binding. As with CLM, large chemical shift changes (⬎0.13 ppm in 1H and 1.1 ppm in 15N) were observed mainly in the loop regions, indicating their involvement in the interaction with fragment III. These changes increased upon continued addition of fragment III. Signals corresponding to Thr63, Arg108, Gly162, and the side chain of Trp163 broadened and disappeared upon the addition of 1 molar equivalent of fragment III. On the basis of the largest observed chemical shift change (100 Hz for the amide proton of Leu109), we determined a lower limit of ⬃600 s–1 for the dissociation rate constant, which corresponds to an estimated Kd of ⬃60 ␮M (assuming kon to be under diffusion control with a value of ⬃107 M⫺1 s⫺1). The titration results suggest that the protein is in intermediate to fast exchange on the NMR time scale in its complex with fragment III, as judged by 1H–15N HSQC cross-peak broadening or repositioning. It is not unreasonable to assume that the natural substrate, CLM, binds more tightly to CalC than the highly truncated fragment III. The 1H and 15N chemical shift changes for backbone amides (Figure 4) define the CalC-binding surface for fragment III/CLM with CalC. Both CLM and fragment III induce similar conformational changes in CalC upon binding as indicated by similar chemical shift changes. Chemical shift perturbations upon binding of CLM/fragment III were found to be concentrated within loop regions L1 (residues 53–70), L2 (residues 79–93), L3 (residues 107–121), and helices ␣2 (residues 146– 156) and ␣3 (residues 159–170). Most of the hydrophobic interactions within the determined CalC–CLM complex involve residues in the loop L1 (Phe53, Pro54, Pro58), helix ␣3 (Met159, Trp163, Pro164, Ile166), and loop L3 (Phe112, Leu109) (Figure 4, panel d). In addition, charged residues such as Arg108, Arg114 from loop L3, Asn60 from loop L1, and Asp160 and Thr165 from ␣3-helix contribute to the electrostatic interactions. The observed NMR shift perturbations upon fragment III/CLM-binding were used as input to HADDOCK (33) to derive a model for the 1:1 CalC–CLM complex. It should also be noted that including the NMR-elucidated interacting residues and the biochemically established CLM www.acschemicalbiology.org

enediyne–Gly113 interaction (19, 20) as an ambiguous interaction restraint (AIRs) in HADDOCK did not significantly alter the final structural model. In the resulting model (Figure 4), the enediyne warhead is oriented toward the neutral loop (L3) and a 26 Å ⫻ 14 Å ⫻ 14 Å hydrophobic tunnel engulfs the CLM aryltetrasaccharide (Figure 4, panel c). A number of intermolecular hydrophobic and hydrogen-bonding interactions account for the specific recognition within this complex (Figure 4, panel d). DNA binding to CalC. There exists no prior evidence for CalC–DNA interaction. However, given that several START domain proteins are either associated with, or directly involved in, transcriptional regulation (31), we used the NMR chemical shift perturbation method described above to test the ability of CalC to bind DNA. Given that there was no a priori knowledge regarding the putative CalC DNA “target”, a structurally characterized double-stranded DNA (dsDNA) 11-mer derived from the Escherichia coli RNA-polymerase promoter region (34) was employed for this initial study. Titration of [U-15N]-CalC with dsDNA, followed by NMR 2D-1H–15N HSQC, identified a set of residue perturbations. These perturbations revealed that CalC binds to the dsDNA probe and localized the CalC–DNA interaction surface to the unique C-terminal helix, ␣2 within CalC (Figure 5). Large chemical shift perturbations were restricted primarily to helix ␣2, (Lys150–Asn157), while residues spatially close to the helix ␣2 (Phe112, Gly113, Arg114, Ile115 from Loop L3, and Tyr28 and Asp29 from the N-terminus) also showed some perturbation in their chemical shift values (Figure 5). Notably, the highfrequency shifts of several backbone nitrogens and amide protons (Asp29, Arg114, Lys150, Lys153, Arg154, and Asn157) are indicative of possible hydrogen bonding to the DNA phosphate backbone (35). From the maximum observed chemical shift change of 225 Hz for amide proton of the residue Lys153, we determined a lower limit of ⬃1410 s⫺1 for the dissociation rate constant, which corresponds to an estimated Kd range of ⬃14–141 ␮M (assuming kon to be under diffusion control with a value of ⬃106–107 M⫺1 s⫺1). The observed changes indicate that the CalC⫺DNA interaction is in fast exchange regime on NMR chemical shift time scale, indicating that the binding is weak. Many proteins that interact with specific DNA sequences bind to nonspecific DNA sequences with weak affinity, and such initial nonspecific interactions play an important VOL.1 NO.7 • 451–460 • 2006

455

Figure 4. NMR chemical shift titration curves for the binding of fragment III to [U-15N]-CalC. a) Changes in chemical shifts upon titration of CalC with fragment III plotted by residue number: black, backbone amide nitrogens; magenta, amide protons. The absence of a bar in the plot indicates the presence of a proline residue or an unmeasured shift due to overlap. b) Regions of the HSQC spectrum displaying changes in cross-peak positions of various residues (Asn60, Leu109, Phe112, and Asp160) upon addition of fragment III at concentrations of 0, 0.1, 0.4, 0.7, 1.1, 1.5, 2.0, and 3.5 mM (decreasing darkness). c) CalC–CLM interacting surface: CalC is represented by the ribbon model, CLM as a stick model; protein residues with significant chemical shift changes in the presence of CLM are colored yellow. d) Stereoview of the CalC–CLM interaction surface.

role in translocation of DNA binding proteins to their target sequence in vivo (36). While the CalC–DNA binding may be tighter to a specific DNA sequence, such specific CalC binding sequences have yet to be determined. The restraints derived from NMR shift perturbations served as input to HADDOCK (33) to derive a model for the CalC–DNA complex. The interaction of positively charged residues from helix ␣2 with the phosphate backbone of the DNA is shown (Figure 5). Mechanistic Implications. To date, the structural elucidation of proteins that sequester highly reactive metabolites capable of DNA damage have revealed the involvement of two major structural folds (37). The first structural fold used for sequestering DNA-damaging metabolites is a dimer of monomers containing two ␤␣␤␤␤-repeats found both within the bleomycin (BLM) and mitomycin (MTM) resistance proteins (BlmA and Mrd, respectively). In both BlmA and Mrd, this ␤␣␤␤␤rich structure provides a concave binding pocket (37). This pocket within BlmA predominately employs electrostatic interactions to bind BLM, while the analogous cavity in Mrd utilizes ␲-stacking interactions of aromatic residues to bind MTM. The second structural fold known to control DNA-damaging natural products is the immunoglobulin fold used by the 9-membered chromoprotein 456

VOL.1 NO.7 • 451–460 • 2006

SINGH ET AL.

enediynes (Figure 1, 7–11). Specifically, stabilizing 9-membered enediyne carrier proteins employ this ␤-sheet-rich fold to provide a hydrophobic pocket that protects the extremely labile enediyne chromophores (37). Distinct from BlmA, Mrd, and enediyne chromoproteins, CalC is a member of the START domain structural superfamily. Thus, this study illuminates, for the first time, the START domain as another fold used by nature to specifically sequester/control highly reactive metabolites capable of DNA damage. The START domain is a conserved protein module that displays remarkable functional versatility and is found in eukaryotes, archaea, and bacteria (30). START domains are involved in diverse cellular functions that include ligand binding [e.g., the START domain of human MLN64, STARD3, which binds to and regulates the transport of cholesterol (38, 39)], catalysis [e.g., the cyclase/ aromatase TcmN in tetracenomycin biosynthesis (40)], and even signal transduction [e.g., the potential tumor suppressor STARD12 (21, 41)]. The most prominent structural feature of the START domain is the presence of a helix-grip fold, which forms a binding pocket for hydrophobic ligands (30). Within CalC, this prominent structural fold serves to bind the CLM aryltetrasaccharide and thereby bury the DNA-recognition unit of CLM. In the www.acschemicalbiology.org

ARTICLE studies of the inactivation of CLM through CalC self-sacrifice and the established minimal enediyne-disaccharide unit required for CalC recognition (19). The demonstration that START domains are efficient scaffolds for CLM-binding may also have broader relevance. For example, in the context of CLM-based therapeutics such as MyloTarg, the current route of CLM intracellular transport from the lysosome to the nucleus (where the enediyne ultimately renders its lethal effects) remains unknown (16, 17). Interestingly, human START domains are broadly distributed within cells, including the nucleus (STARD4, D5, D6, and D10), cytosol (STARD2, D4, D5, D11, D13, and D15), mitochondria (STARD10), Golgi (STARD11), plasma membrane (STARD12), and even endosomes (STARD3) (21). Therefore, the present study, which reveals the START domain as an efficient enediynebinding scaffold, presents a compelling basis from which to propose that START domain proteins may also serve to chaperone and/or mediate the requisite intracellular transport and translocation of CLM within human cells. CLM-induced DNA strand scission is initiated by reductive activation. For example, in the presence of glutathione (GSH), CLM initially binds DNA reversibly, and the DNAFigure 5. NMR chemical shift titration curves for the binding of DNA bound CLM reacts with GSH to form [U-15N]-CalC. a) Changes in chemical shifts upon titration of CalC with GSH–CLM disulfide as the major product (9). DNA, (blue) backbone amide nitrogen and (magenta) amide proton, A number of long-lived, DNA-free intermediplotted as a function of the residue number. The absence of a bar in ates exist en route to this key GSH–CLM the plot indicates the presence of a proline residue or an unmeasured disulfide, which can also dissociate from shift due to overlap. b) Regions of HSQC spectra displaying changes in the positions of cross peaks corresponding to various residues (Asp29, DNA. The GSH–CLM disulfide intermediate Arg114, Lys153) at DNA concentrations of 0, 0.05, 0.08, and 0.1 mM ultimately leads to the hetero-Michael (decreasing darkness). c) CalCⴚDNA interaction surface: CalC is repreadduct (Figure 1, dihydrothiophene I), which sented by the blue ribbon; residues exhibiting significant chemical rapidly cycloaromatizes to give the reactive shift perturbations are shown in red. d) CalC residues exhibiting the 1,4-diradical species required for hydrogen largest chemical shift perturbations upon titration with DNA. abstraction from the DNA backbone (10, 42, CalC–CLM complex, the trisulfide trigger of CLM is 43). The current structural study revealed a minimum solvent-exposed to facilitate activation, while the eneCalC–fragment III association of 104 M⫺1, and efforts to diyne warhead is positioned for Gly113 ␣-hydrogen elucidate an association for CalC–CLM were prohibited abstraction to initiate the proteolytic CalC self-sacrifice by CLM solubility. Given the CLM–DNA association is event. Thus, the structure of the CalC–CLM complex is estimated to be 106–108 M⫺1 (44, 45) and in vitro CLM inactivation via CalC self-sacrifice is favored even in the consistent with the previous in-depth mechanistic www.acschemicalbiology.org

VOL.1 NO.7 • 451–460 • 2006

457

presence of excess DNA (19, 20), we conclude the estimated fragment III–CalC weak association may not reflect the true CalC–CLM binding affinity. The current NMR study also clearly divulged, for the first time, evidence for association of CalC with DNA. In the context of “self-sacrifice”, the apparent localization of CalC to the intended enediyne target (DNA) in conjunction with an apparent affinity of CalC for CLM is consistent with the demonstrated efficient capture and inactivation

of CLM by CalC to ultimately prevent DNA damage. Given that the interaction of many regulatory DNA-binding proteins with their target sites is usually preceded by binding to nonspecific DNA, the current study does not support or refute the existence of specific CalC-binding sequences. It will be of interest in future studies to determine whether CalC displays sequence specificity and whether the observed CalC–DNA interaction offers additional direct or indirect effects within the CLM producing bacterium.

METHODS

NaCl, pH 7.3 (A280 detection, 0.5 mL min⫺1). CalC-containing fractions (67–85 mL) were collected, concentrated, and stored in 5 mM BisTris, 100 mM NaCl, 10 mM MgCl2, pH 7.3 (NMR buffer). Generation of CLM Fragment III. Fragment III (Figure 1) was produced by refluxing 10 mg of CLM ␣3I in 10 mL of wet acetone with pyridinium p-toluene-sulfonate (0.1 equiv) at 70 °C. The hydrolysis was monitored by HPLC using a reverse-phase column (Phenomenex Luna C18, 250 ⫻ 4.6 mm) with a gradient of 10%–90% acetonitrile in H2O over 20 min at a flow rate of 1 mL min⫺1. The products were detected under UV-280 nm by a PDA detector with a retention time for CLM ␣3I of 15.5 min and for fragment III of 11.6 min. After 20 h, solvent was evaporated, and the fragment III was purified by preparative HPLC isolation using a reverse-phase column (DiscoveryBIO C18, 25 cm ⫻ 21.2 mm, 10 ␮m, Supelco) with a gradient of 10– 90% acetonitrile in H2O over 20 min at a flow rate of 10 mL min⫺1. After lyophilization, 0.8 mg of fragment III was obtained (8% yield). The product was confirmed by 1H NMR on Varian UNITY INOVA 500 MHz using a CapNMR probe. Binding Studies. For CLM-binding studies, CLM was dissolved in 45–50 ␮L of DMSO to give a stock solution of 15–20 mM CLM. 2D 1 H–15N HSQC spectra of CalC (100 ␮M) were recorded in NMR buffer with the successive addition of increasing CLM at 30 °C. The 2D 1H–15N-HSQC of 100 ␮M CalC in NMR buffer containing 15% DMSO was used as a control. For fragment III-binding studies, fragment III was dissolved in NMR buffer to give a stock solution of 15 mM. The 2D 1H–15N HSQC spectra of 400 ␮M CalC with 0.14, 0.57, 1.0, 1.57, 2.14, 2.85, and 5.0 equiv of fragment III were recorded at 30 °C. For CalC–DNA studies, the DNA employed was dsDNA: 1, 5=-GCATATGATAG and 5=-CTATCATATGC and the 2D 1H–15N HSQC spectra of CalC (100 ␮M) recorded in NMR buffer with 0.05, 0.21, 0.42, 0.84, and 1.26 equiv of DNA at 30 °C. All 1H–15N HSQC spectra were recorded using a time domain data size of 400 t1, 1596 t2 complex points, and 16 transients per t1 increment. NMR Spectra Used for the Structure Determination. NMR experiments were carried out at 30 °C on Varian UNITYINOVA 900 MHz, 800 MHz, and 600 MHz spectrometers equipped with a 1H-13C-15N triple-resonance cold probe (800 MHz, 600 MHz) with a 5 mm z-shielded gradient or a RT probe (900 MHz) with a 5 mm x, y, z-shielded gradient. For the backbone resonance assignments, (3D) HNCO, HNCACB, CBCA(CO)NH, NOESY-(15N,1H)-HSQC spectra were recorded as previously described (47), and for the side chain resonance assignments, 3D HBHA(CO)NH, H(CCO)NH, C(CO)NH, H(C)CH TOCSY, and (H)CCG TOCSY data sets were recorded. NOE distance constraints were obtained from 3D-NOESY(15N,1H)-HSQC, and 3D NOESY-(13C,1H)-HSQC spectra with 100 ms mixing times. Spectra were processed with NMRPipe (48) and analyzed using NMRView (49) software packages. Chemical Shift-Derived and Hydrogen Bond Restraints. Assigned chemical shifts were determined for 97% of the nuclei (BioMagResBank accession code 6726). The 13C␣, 13C␤, 13Co, 1H␣, and 15N chemical shifts of the assigned residues served as input for

Cloning, Expression, and in Vivo Assays. The N-terminal truncation expression constructs of CalC were amplified from pJB2011 using the following primer pairs: CalC27-181, 5=-AAGCCGAAGCATATGAACTACGACCCGTTC-3=/5=-ATATATAAGCTTTCACTTCTTCGCCCCTTCC-3=; CalC1-112, 5=-ATATATCATATGCATCACCATCACCATCACACTCAGGAGAAGACCGCA-3=/5=-CGGGTCAAGCTTTCAGAAGCCGTTGAGCCG-3=; CalC27-112, 5=-ATATATCATATGCATCACCATCACCATCACAACTACGACCCGTTCGTC-3=/5=-CGGGTCAAGCTTTCAGAAGCCGTTGAGCCG-3=; and CalC114-181, 5=-AACGGCCATATGCGGATCGACCCGGAC-3=/5=-ATATATAAGCTTTCAGTGATGGTGATGGTGATGCTTCTTCGCCCCTTCCTC-3=. After restriction digestion with NdeI/ HindIII (Promega), the PCR products were ligated into NdeI/HindIIIlinearized pET21a (Novagen) to yield constructs pMH16 (CalC27– 181), pMH20 (CalC1–112), pMH21 (CalC27–112), and pMH22 (CalC114–181), respectively. Each expression plasmid was confirmed by sequencing and subsequently transformed into E. coli strain BL21(DE3)Gold (Stratagene) for expression at 37 °C. In vivo CLM resistance assays for CalC, CalC27–181, CalC1–112, CalC27– 112, and CalC114–181 were performed on LB-agar plates supplemented with 75 ␮g mL⫺1 carbenicillin, 1 mM isopropyl-␤-Dthiogalactopyranoside, and 30 ng mL–1 CLM as previously described (19). In this study, cells containing the CalC expression construct, as well as control cells containing empty vector, were grown to an o.d. of ⬃2, and an equal cell density from each starter culture was plated. Plates were incubated for 12 h at 37 °C, and the clones were assessed for survival. Labeling. Labeling of CalC27–181, CalC1–112, CalC27–112, and CalC114–181 with 15N and double-labeling of CalC27–181 with 13C and 15N was achieved by growth of BL21(DE3)Gold transformed with pMH16, pMH20, pMH21, and pMH22 in modified minimal medium according to Zhao et al. (46). The minimal salts medium for 15N/13C-labeling was supplemented with 4 g of 13Cglucose L–1 and adjusted to pH 7.2. Protein expression was performed in 2.5 L Fernbach flasks, and cells were grown for 14 h at 37 °C to an o.d. of 4.8. Cells were harvested by centrifugation at 3000g for 20 min. Protein Purification. Cells were resuspended in binding buffer (50 mM sodium phosphate, 25 mM NaCl, pH 6.8) and disrupted in a pressure cell (Thermo Electron) at 12,000 psi. Insoluble debris was removed by centrifugation at 10,000g for 1 h, and the supernatant was loaded onto a HiTrap SP FF column (Amersham Biosciences) for ion exchange chromatography using a NaCl-gradient (50 mM sodium phosphate, pH 6.8, 25 mM–1 M NaCl over 41 mL, A280 detection, 0.5 mL min⫺1). Under these conditions, the desired protein eluted at 175 mM NaCl, and CalC-containing fractions were collected and concentrated by ultrafiltration (10,000 MWCO-membrane, Centriprep and Centricon YM10, Millipore). Concentrated protein was loaded onto a HiPrep 16/60 Sephacryl S-200 HR column (Amersham Biosciences) for gel filtration in 50 mM BisTris, 200 mM

458

VOL.1 NO.7 • 451–460 • 2006

SINGH ET AL.

www.acschemicalbiology.org

ARTICLE the TALOS program (26). For better convergence, a number of hydrogen bond restraints were introduced for the backbone amide protons on the basis of amide 1H-2H exchange results, 13C␣-/13C␤ secondary shifts, and NOE data. Hydrogen bonds were enforced by using the following restraints: 1.8–2.3 Å for d(N-H, O) and 2.7–3.3 Å for d(N, O). The hydrogen bonds between N–Hi and O–Cj in the ␤-sheet structures were included as restraints only if the ␤-sheet interstrand dNN (i, j), and d␣N (i, j) NOE cross peaks were observed. Hydrogen bond constraints for ␣-helices were included when NOEs corresponding to the secondary structure d␣N (i, i⫹3) for ␣-helices were observed. Structure Calculations. Structure calculations (50) were performed with CNS (24) using the ARIA setup and simulated annealing protocols (25). The best 20 structures out of 100 calculated were selected on the basis of lowest total energy. PROCHECK-NMR (51) was used to assess the quality of the final ensemble of conformers. Analysis of the final 20 structures showed that 93.3% were in the favored region of the Ramachandran plot. Structures were visualized with the programs MOLMOL (52) or PyMOL (www.pymol.org). Modeling the CalC–CLM and CalC–DNA Complexes. Coordinates of CLM were taken from the NMR structure of the CLM–DNA complex (2PIK) (53). Topology and parameter files were generated by the PRODRG server (54). The experimentally determined distance and dihedral restraints for CalC (Supplementary Table 1) were applied in a simulated annealing protocol using CNS and HADDOCK (33). AIRs were generated based on the chemical shift mapping data of CalC with fragment III. “Active” and “passive” residues were distinguished on the basis of their amount of chemical shift perturbation; in the NMR titration data, active residues correspond to all residues showing a significant chemical shift perturbation (⬎0.03 ppm for 1H and ⬎0.3 ppm for 15N) upon complex formation. From the list of active and passive residues, 24 AIRs were defined with an upper distance boundary of 3.5 Å. A total of 500 conformers of the CalC⫺CLM complex was generated using only the AIRs, van der Waals energy, and electrostatic terms in CNS. The 100 conformers with lowest molecular energies were subsequently subjected to semiflexible simulated annealing and refinement with explicit water and only backbone restraints for residues outside the interface. Residues 56⫺66, 79–92, 106–119, 130–144, and 150–168 of CalC, and full CLM were allowed to be flexible in all stages of the docking procedure. After the calculation, structures were ranked according to their intermolecular energy (the sum of electrostatic, van der Waals, and AIR energy terms). Ten structures with low restraint energy were accepted as a final structure. For the CalC–DNA complex, the DNA coordinates were taken from the DNA structure 1SKP, d(gcatatgatag).d(ctatcatatgc), a consensus sequence for promoters recognized by sigma-k RNA polymerase (34). Fourteen AIRs were defined with an upper distance boundary of 3.5 Å. A total of 20 conformers of the CalC–DNA complex was generated using only the AIRs, van der Waals energy, and electrostatic terms in CNS. The 10 conformers with lowest molecular energies were subsequently subjected to semiflexible simulated annealing and refinement with explicit water and only backbone restraints for residues outside the interface. Residues 28⫺32, 62–65, 89–93, 109–116, and 148–155 of CalC, and the middle four base pairs in DNA were allowed to be flexible in all stages of the docking procedure. After the calculation, structures were ranked according to their intermolecular energy (the sum of electrostatic, van der Waals, and AIR energy terms). The structure with low restraint energy was accepted as a final structure. Accession Codes. The chemical shifts of CalC at pH 7.3 and 30 °C have been deposited in the BioMagResBank under accession number 6726. The atomic coordinates of the ensemble of 20 structures that represent the solution structure of CalC, together with the complete list of constraints used for the final structure calculation, have been deposited in the Protein Data Bank (PDB), under accession number 1ZXF. Atomic coordinates of the ensemble of 10 struc-

www.acschemicalbiology.org

tures that represent the model of the complex of CalC–CLM and a minimum restraint energy of complex of CalC–DNA, along with the complete list of AIR restraints derived from chemical shift perturbations, have been deposited in the PDB, under accession number 2GKC and 2GKD, respectively. Acknowledgments: This work was supported in part by the National Institutes of Health grants CA84374, AI52218, and GM70637, and a NCDDG grant from the National Cancer Institute (U19 CA113297). M. H. Hager is a postdoctoral fellow of the German Academy of Scientists Leopoldina (BMBF-LPD 9901/882) and J. S. Thorson is a Romnes Fellow. NMR data were collected at the National Magnetic Resonance Facility at Madison, which is supported in part by grants P41 RR02301, P41 GM66326, the National Institutes of Health, and a Protein Structure Initiative through Grant P50 GM64598. We gratefully thank Wyeth Research for providing the parent enediynes and M. Tonelli from NMRFAM for providing dsDNA for binding studies. Supporting Information Available: This material is available free of charge via the Internet.

REFERENCES 1. Jones, G. B., and Fouad, F. S. (2002) Designed enediyne antitumor agents, Curr. Pharm. Des. 8, 2415–2440. 2. Danishefsky, S. J., and Shair, M. D. (1996) Observations in the chemistry and biology of cyclic enediyne antibiotics—total syntheses of calicheamicin and dynemicin, J. Org. Chem. 61, 16–44. 3. Smith, A. L., and Nicolaou, K. C. (1996) The enediyne antibiotics, J. Med. Chem. 39, 2103–2117. 4. Davies, J., Wang, H., Taylor, T., Warabi, K., Huang, X. H., and Andersen, R. J. (2005) Uncialamycin, a new enediyne antibiotic, Org. Lett. 7, 5233–5236. 5. Thorson, J. S., Shen, B., Whitwam, R. E., Liu, W., Li, Y., and Ahlert, J. (1999) Enediyne biosynthesis and self resistance. A Progress Report, Bioorg. Chem. 27, 172–88. 6. Thorson, J. S., Sievers, E. L., Ahlert, J., Shepard, E., Whitwam, R. E., Onwueme, K. C., and Ruppen, M. (2000) Understanding and exploiting nature’s chemical arsenal: the past, present and future of calicheamicin research, Curr. Pharm. Des. 18, 1841–1879. 7. Oku, N., Matsunaga, S., and Fusetani, N. (2003) Shishijimicins A-C, novel enediyne antitumor antibiotics from the ascidian Didemnum proliferum(1), J. Am. Chem. Soc. 125, 2044–2045. 8. Zein, N., Sinha, A. M., McGahren, W. J., and Ellestad, G. A. (1988) Calicheamicin gamma 1I: an antitumor antibiotic that cleaves double-stranded DNA site specifically, Science 240, 1198–1201. 9. Myers, A. G., Cohen, S. B., and Kwon, B. M. (1994) A study of the reaction of calicheamicin gamma(1) with glutathione in the presence of double-stranded DNA, J. Am. Chem. Soc. 116, 1255–1271. 10. DeVoss, J. J., Hangeland, J. J., and Townsend, C. A. (1990) Characterization of the in vitro cyclization chemistry of calicheamicin and its relation to DNA cleavage, J. Am. Chem. Soc. 112, 4554–4556. 11. Kappen, L. S., and Goldberg, I. H. (1993) DNA conformationinduced activation of an enediyne for site-specific cleavage, Science 261, 1319–1321. 12. Watanabe, C. M. H., Supekova, L., and Schultz, P. G. (2002) Transcriptional effects of the potent enediyne anti-cancer agent Calicheamicin gamma (I)(1), Chem. Biol. 9, 245–251. 13. Stassinopoulos, A., Ji, J., Gao, S., and Goldberg, I. H. (1996) Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog, Science. 272, 1943–1946. 14. Damle, N. K. (2004) Tumour-targeted chemotherapy with immunoconjugates of calicheamicin, Exp. Opin. Biol. Ther. 4, 1445–1452. 15. Abe, S., and Otsuki, M. (2002) Styrene maleic acid neocarzinostatin treatment for hepatocellular carcinoma, Curr. Med. Chem. 2, 715–726. VOL.1 NO.7 • 451–460 • 2006

459

16. Jedema, I., Barge, R. M., van der Velden, V. H, Nijmeijer, B. A., van Dongen, J. J, Willemze, R., and Falkenburg, J. H. (2004) Internalization and cell cycle-dependent killing of leukemic cells by Gemtuzumab Ozogamicin: rationale for efficacy in CD33-negative malignancies with endocytic capacity, Leukemia 18, 316–325. 17. Wu, A. M., and Senter, P. D. (2005) Arming antibodies: prospects and challenges for immunoconjugates, Nat. Biotechnol. 23, 1137–1146. 18. Ahlert, J., Shepard, E., Lomovskaya, N., Zazopoulos, E., Staffa, A., Bachmann, B. O., Huang, K., Fonstein, L., Czisny, A., Whitwam, R. E., Farnet, C. M., and Thorson, J. S. (2002) The calicheamicin gene cluster and its iterative type I enediyne PKS, Science 297, 1173–1176. 19. Biggins, J. B., Onwueme, K. C., and Thorson, J. S. (2003) Resistance to enediyne antitumor antibiotics by CalC self-sacrifice, Science 301, 1537–1541. 20. Biggins, J. B., Prudent, J. R., Marshall, D. J., Ruppen, M., and Thorson J. S. (2000) A continuous assay for DNA cleavage: the application of “break lights” to enediynes, iron-dependent agents, and nucleases, Proc. Natl. Acad. Sci. U.S.A. 25, 13537–13542. 21. Alpy, F., and Tomasetto, C. (2005) Give lipids a START: the StARrelated lipid transfer (START) domain in mammals, J. Cell. Sci. 118, 2791–2801. 22. Soccio, R. E., and Breslow, J. L. (2003) StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism, J. Biol. Chem. 278, 22183–22186. 23. McGuffin, L. J., Bryson, K., and Jones, D. T. (2000) The PSIPRED protein structure prediction server, Bioinformatics 16, 404–405. 24. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr., D: Biol. Crystallogr. 54, 905–921. 25. Linge, J. P., Habeck, M., Rieping, W., and Nilges, M. (2003) ARIA: automated NOE assignment and NMR structure calculation, Bioinformatics 19, 315–316. 26. Cornilescu, G., Delaglio, F., and Bax, A. (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology, J. Biomol. NMR 13, 289–302. 27. Holm, L., and Sander, C. (1993) Protein structure comparison by alignment of distance matrices, J. Mol. Biol. 233, 123–138. 28. Gibrat, J. F., Madej, T., and Bryant, S. H. (1996) Surprising similarities in structure comparison, Curr. Opin. Struct. Biol. 6, 377–385. 29. Gajhede, M., Osmark, P., Poulsen, F. M., Ipsen, H., Larsen, J. N., Joost van Neerven, R. J., Schou, C., Lowenstein, H., and Spangfort, M. D. (1996) X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy, Nat. Struct. Biol. 3, 1040–1045. 30. Iyer, L., Koonin, E., and Aravind, L. (2001) Adaptations of the helixgrip fold for ligand binding and catalysis in the START domain superfamily, Proteins: Struct., Funct., Genet. 43, 134–144. 31. Ponting, C. P., and Aravind, L. (1999) START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins, Trends. Biochem. Sci. 24, 130–132. 32. Dekker, N., Cox, M., Boelens, R., Verrijzer, C. P., van der Vliet, P. C., and Kaptein, R. (1993) Solution structure of the POU-specific DNAbinding domain of Oct-1, Nature 362, 852–855. 33. Dominguez, C., Boelens, R., and Bonvin, A. M. (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information, J. Am. Chem. Soc. 125, 1731–1737. 34. Tonelli, M., Ragg, E., Bianucci, A. M., Lesiak, K., and James, T. L. (1998) Nuclear magnetic resonance structure of d(GCATATGATAG). d(CTATCATATGC): a consensus sequence for promoters recognized by ␴K RNA polymerase, Biochemistry 37, 11745–11761.

460

VOL.1 NO.7 • 451–460 • 2006

SINGH ET AL.

35. Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H., and Morikawa, K. (2000) Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer, Proc. Natl. Acad. Sci. U.S.A. 97, 8257–8262. 36. Jen-Jacobson, L. (1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state, Biopolymers 44, 153–180. 37. Galm, U., Hager, M. H., Van Lanen, S. G., Ju, J., Thorson, J. S., and Shen, B. (2005) Antitumor antibiotics: bleomycin, enediynes, and mitomycin, Chem. Rev. 105, 739–758. 38. Tsujishita, Y., and Hurley, J. H. (2000) Structure and lipid transport mechanism of a StAR-related domain, Nat. Struct. Biol. 7, 408–414. 39. Romanowski, M. J., Soccio, R. E., Breslow, J. L., and Burley, S. K. (2002) Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain, Proc. Natl. Acad. Sci. U.S.A. 99, 6949–6954. 40. Thompson, T. B., Katayama, K., Watanabe, K., Hutchinson, C. R., and Rayment, I. (2004) Structural and functional analysis of tetracenomycin F2 cyclase from Streptomyces glaucescens. A type II polyketide cyclase, J. Biol. Chem. 279, 37956–37963. 41. Homma, Y., and Emori, Y. (1995) A dual functional signal mediator showing RhoGAP and phospholipase C-delta stimulating activities, EMBO J. 14, 286–291. 42. Chatterjee, M., Cramer, K. D., and Townsend, C. A. (1993) Kinetic nature of thiol activation in DNA cleavage by calicheamicin, J. Am. Chem. Soc. 115, 3374–3375. 43. Kuduvalli, P. N., Townsend, C. A., and Tullius, T. D. (1995) Cleavage by calicheamicin ␥1I of DNA in a nucleosome formed on the 5S RNA gene of Xenopus borealis, Biochemistry 34, 3899–3906. 44. Drak, J., Iwasawa, N., Danishefsky, S., and Crothers, D. M. (1991) The carbohydrate domain of calicheamicin gamma I1 determines its sequence specificity for DNA cleavage, Proc. Natl. Acad. Sci. U.S.A. 88, 7464–7468. 45. Ding, W. D., and Ellestad, G. A. (1991) Evidence for hydrophobic interaction between calicheamicin and DNA, J. Am. Chem. Soc. 113, 6617–6620. 46. Zhao, Q., Frederick, R., Seder, K., Thao, S., Sreenath, H., Peterson, F., Volkman, B. F., Markley, J. L., and Fox, B. G. (2004) Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N, J. Struct. Funct. Genomics 5, 87–93. 47. Palmer, A. G., III, Fairbrother, W. J., Cavanagh, J., Wright, P. E., and Rance, M. (1992) Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins, J. Biomol. NMR 2, 103–108. 48. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes, J. Biomol. NMR 6, 277–293. 49. Johnson, B. A. (2004) Using NMRView to visualize and analyze the NMR spectra of macromolecules, Methods. Mol. Biol. 278, 313–352. 50. Bonvin, A. M., Boelens, R., and Kaptein, R. (2005) NMR analysis of protein interactions, Curr. Opin. Chem. Biol. 9, 501–508. 51. Laskowski, R. A., Moss, D. S., and Thornton, J. M. (1993) Mainchain bond lengths and bond angles in protein structures, J. Mol. Biol. 231, 1049–1067. 52. Koradi, R., Billeter, M., and Wuthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures, J. Mol. Graphics 14, 51–55. 53. Kumar, R. A., Ikemoto, N., and Patel, D. J. (1997) Solution structure of the calicheamicin gamma 1I-DNA complex, J. Mol. Biol. 265, 187–201. 54. Schuttelkopf, A. W., and van Aalten, D. M. (2004) PRODRG: a tool for high-throughput crystallography of protein-ligand complexes, Acta. Crystallogr., D: Biol. Crystallogr. 60, 1355–1363.

www.acschemicalbiology.org