Solution Structure of the Covalent Duocarmycin A-DNA Duplex Complex

JMB—MS 450 Cust. Ref. No. PEW 182/94-Rev

[SGML] J. Mol. Biol. (1995) 248, 162–179

Solution Structure of the Covalent Duocarmycin A–DNA Duplex Complex Chin Hsiung Lin and Dinshaw J. Patel* Cellular Biochemistry and Biophysics Program Memorial Sloan-Kettering Cancer Center, New York NY 10021, U.S.A.

*Corresponding author

Duocarmycin A is an antitumour antibiotic that binds covalently to the minor groove N-3 position of adenine with sequence specificity for the 3'-adenine in a d(A-A-A-A) tract in duplex DNA. The adenine ring becomes protonated on duocarmycin adduct formation resulting in charge delocalization over the purine ring system. We report on the solution structure of duocarmycin A bound site specifically to A12 (designated *A12+ ) in the sequence context d(T3-T4-T5-T6)·d(A9-A10-A11-*A12+ ) within a hairpin duplex. The solution structure was solved based on a combined NMR-molecular dynamics study including NOE based intensity refinement. The A and B-rings of duocarmycin are positioned deep within the walls of the minor groove with the B-ring (which is furthest from the covalent linkage site) directed towards the 5'-end of the modified strand. Duocarmycin adopts an extended conformation and is aligned at 045° to the helix axis with its non-polar concave edges interacting with the floor of the minor groove while its polar edges are sandwiched within the walls of the minor groove. The T3·*A12+ modification site pair forms a weak central Watson-Crick hydrogen bond in contrast to all A·T and G·C pairs, which align through standard Watson-Crick pairing in the complex. The helical parameters are consistent with a minimally perturbed right-handed duplex in the complex with minor groove width and x-displacement parameters indicative of a B-form helix. A striking feature of the complex is the positioning of duocarmycin A within the walls of the minor groove resulting in upfield shifts of the minor groove sugar protons, as well as backbone proton and phosphorus resonances in the DNA segment spanning the binding site. Keywords: covalent duocarmycin–DNA complex; solution structure; duocarmycin alignment and directionality; intermolecular interactions in the minor groove

Introduction Duocarmycin A (Structure 1) is a potent natural antitumor antibiotic produced by Streptomyces sp which is effective against murine lymphocytic leukemia P388 and sarcoma 180 in mice (Takahashi et al., 1988; Ichimura et al., 1991). The reaction between duocarmycin A and DNA was reported to be similar to that of the related antibiotic (+)-CC-1065 (Structure 2) with DNA (Sugiyama et al., 1990; Boger et al., 1990). This family of antibiotics covalently binds to N-3 of adenine in duplex DNA with the drug positioned in the minor groove and directed Abbreviations used: NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; r.m.s.d., root-mean-square deviation.

0022–2836/95/160162–18 $08.00/0

towards the 5' side of the covalent modification site (reviewed by Warpehoski & Hurley, 1988). Complex formation generates an extra positive charge which is delocalized over the modified adenine ring (Structure 3) in these covalent drug–adenine complexes at the duplex level (Lin & Hurley, 1990; Sugiyama et al., 1990). Duocarmycin covalently binds with high specificity to adenine in sequences 5'-d(A/T-A-A-*A)-3' and 5'-d(A/T-T-T-*A-Pu)-3', where *A indicates the covalent modification site (Boger et al., 1990; 1991a,b). A comprehensive study of the DNA binding properties of duocarmycins, their unnatural enantiomers and their alkylation containing subunit fragments have led to comparative models for the alignment of duocarmycins and their analogs in the minor groove of duplex DNA (Boger et al., 1994). Our laboratory has set out to define and compare the 7 1995 Academic Press Limited

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Duocarmycin–DNA Complex

Structure 1

structures of duocarmycin A–DNA complexes at the duplex and triplex level with a preliminary report on structural aspects of the latter complex reported previously (Lin & Patel, 1992). The research at the duplex level has been conducted on a hairpin sequence which contains a 5'-A-A-A-A-3' sequence in the stem of the hairpin. We have generated and purified a single covalent duocarmycin adduct at position A12 on the hairpin duplex (Structure 4). A combined NMR-molecular dynamics approach including intensity based refinement has been applied to define the three-dimensional structure of the duocarmycin A–hairpin duplex complex in solution. The resulting structure defines the alignment of duocarmycin in the minor groove of the DNA and identifies the intermolecular contacts that stabilize the complex.

Results Exchangeable nucleic acid protons in complex All seven imino protons are well resolved in the duocarmycin–hairpin duplex complex and have been assigned following analysis of the NOESY (170 ms mixing time) spectrum of the duocarmycin A–hairpin duplex complex in H2 O buffer (pH 7.0) at 15°C. The imino proton connectivities can be traced in expanded NOESY contour plots between adjacent base-pairs from G8 at one end to G14 at the other end without interruption in the hairpin duplex complex (Figure 1C). These imino proton assignments in the complex and related ones in the control hairpin duplex establish that the imino proton of T3 shifts upfield by 2.0 ppm and that of T4 shifts downfield by

Structure 2

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Duocarmycin–DNA Complex

Structure 3

0.87 ppm on complex formation. The expanded NOESY contour plot outlining the NOE connectivities between the imino protons and the base and amino protons in Figure 1A establishes Watson-Crick base-pairing at C1·G14, C2·G13 and C7·G8 basepairs (guanine imino to cytosine amino proton NOEs across the pair) and at T4·A11, T5·A10 and T6·A9 base-pairs (thymine imino to adenine H2 and amino proton NOEs across the pair). Duocarmycin covalent binding to A12 The imino proton of T3 exhibits NOEs to the H2 proton of A12 (peak A, Figure 1A), as well as to the amino protons of A12 at 8.62 and 8.98 ppm (peaks B and C, Figure 1B) in the duocarmycin A–hairpin duplex complex. These latter amino protons of A12 also exhibit NOEs to the imino proton of T4 (peaks F and G, Figure 1B) in one direction and to the imino proton of G13 (peaks D and E, Figure 1B) in the other direction. The downfield shift of the amino protons of A12 require protonation of this covalently modified adenine ring (4) in the duocarmycin– hairpin duplex complex. There are two NMR parameters for the T3·*A12+ pair (the asterisk stands for the modification site) in the duocarmycin A–hairpin duplex complex that are of interest. One is the 2.0 ppm upfield shift of the

imino proton of T3 on complex formation and the second is the weaker intensity between the imino proton of T3 and the H2 proton of A12 (peak A, Figure 1A) for the T3·*A12+ pair relative to the corresponding NOEs across the other T·A basepairs (peaks B, C and D, Figure 1A). These two observations require that the Watson-Crick hydrogen-bonding between T3 and its protonated partner *A12 is weaker than a standard Watson-Crick T·A pair in the duocarmycin A–hairpin duplex complex. The exchangeable nucleic acid proton chemical shifts in the duocarmycin A–hairpin duplex complex in H2 O buffer at 15°C are listed in Table 1. The complexation chemical shifts on proceeding from the control hairpin duplex to the duocarmycin A–hairpin duplex complex are also listed (in parentheses) in Table 1. Exchangeable duocarmycin protons in the complex A narrow exchangeable resonance is detected at 11.12 ppm in the spectrum of the duocarmycin A–hairpin duplex complex in H2 O buffer. It is assigned to the 1'-NH proton of duocarmycin A based on the observed NOEs to the H3' (peak Q, Figure 1A) and H7 (peak R, Figure 1A) protons of the drug. The duocarmycin 1-NH exchangeable proton

Structure 4

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165

Figure 1. Expanded NOESY contour plots (170 ms mixing time) of the duocarmycin A–hairpin duplex complex in H2 O buffer (pH 7.0) at 15°C. A, The imino proton (11.0 to 15.0 ppm) to the amino, base, and sugar proton (5.0 to 8.3 ppm) region. The intermolecular NOEs between the drug and DNA protons are boxed. The NOE crosspeaks A to V are assigned as follows: A: T3(NH3)A12(H2); B: T4(NH3)-A11(H2); C: T5(NH3)-A10(H2); D: T6(NH3)A9(H2); E: G8(NH1)-C7(NH2e ); F: G8(NH1)-C7(NH2b ); G: G14(NH1)C1(NH2e ); H: G14(NH1)-C1(NH2b ); I: G13(NH1)-C2(NH2e ); J: G13(NH1)-C2(NH2b ); K: T5(NH3)Duo(H3'); L: T4(NH3)-Duo(H5A/ H4a); M: T5(NH3)-Duo(H5A/H4a); N: T3(NH3)-Duo(H5A/H4a); O: Duo(1'-NH)-T6(H1'); P: Duo(1'-NH)A11(H1'); Q: Duo(1'-NH)-Duo(H3'); R: Duo(1'-NH)-Duo(H7); S: T5(NH3)-Duo(H5'); T: T4(NH3)A12(H2); U: T5(NH3)-A11(H2); V: T6(NH3)-A10(H2). B, NOE crosspeaks between the exchangeable protons (8.5 to 15.0 ppm) and the amino protons of *A12 (8.5 to 9.5 ppm). The NOE crosspeaks A to G are assigned as follows: A: *A12(NH2b )-*A12(NH2e ); B: *A12(NH2e )-T3(NH3); C: *A12(NH2b )T3(NH3); D: *A12(NH2e )-G13(NH1); E: *A12(NH2b )-G13(NH1); F: *A12(NH2e )-T4(NH3); G: *A12(NH2b )T4(NH3). The covalently modified protonated adenine A12 is designated by an asterisk. C, The symmetrical imino proton (11.0 to 15.0 ppm) region. The NOE crosspeaks A to F are assigned as follows: A: T3(NH3)-G13(NH1); B: T3(NH3)-T4(NH3); C: T4(NH3)T5(NH3); D: T5(NH3)-T6(NH3); E: T6(NH3)-G8(NH1); F: G13(NH1)G14(NH1).

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resonates at 4.41 ppm while we have been unable to detect the duocarmycin 8-OH exchangeable proton in the complex.

widths in right-handed duplexes. These cross-strand NOEs are detected between the H2 protons of A10, A11 and A12, and the sugar H1' protons of T6, T5 and T4, respectively (peaks I, H and N, respectively, Figure 2A) with intensities comparable to same strand purine H8 to sugar H1' NOEs (Figure 2A) in the duocarmycin A–hairpin duplex complex.

Non-exchangeable nucleic acid protons in the complex An expanded NOESY contour plot (250 ms mixing time) of the base (6.8 to 8.3 ppm) to the sugar H1' (5.0 to 6.4 ppm) proton region of the duocarmycin A–hairpin duplex complex in 2H2 O buffer at 25°C is plotted in Figure 2A. The NOE connectivities between the base and its own and 5'-flanking sugar H1' protons can be traced from C1 to C7 and G8 to G14 along both strands of the stem region of the hairpin duplex complex (Figure 2A). The same connectivities can also be traced in the T5 hairpin loop (designated 3'-Ta-Tb-Tc-Td-Te-5') with weak crosspeaks observed at the Ta-Tb and Tb-Tc steps (Figure 2A). The other regions of the NOESY contour plot have also been analyzed to yield the non-exchangeable base and sugar proton assignments in the duocarmycin A–hairpin duplex complex at 25°C (Table 2). Several non-exchangeable protons exhibit unusual chemical shifts in the duocarmycin A–hairpin duplex complex (Table 2). Thus, the minor groove sugar H1' protons of A11 (5.35 ppm), *A12+ (5.32 ppm) and T6 (5.20 ppm), and to a lesser extent A9 (5.77 ppm), are shifted to high field in the complex (Figure 2A, Table 2). Similarly, the minor groove sugar H4' protons of A11, *A12+ and G13 on the modified strand and of T4, T5, T6 and C7 on the unmodified strand are shifted to high field in the complex (Table 2). Upfield shifts are also observed at the H5',5" protons of T5, T6, *A12+ and G13 in the complex (Table 2). By contrast, the H8 (8.18 ppm) and H2 (8.02 ppm) of the protonated *A12+ modification site residue resonates to lowest field in the complex (Table 2) most likely reflecting protonation of this residue on complex formation. The strength of the cross-strand NOE between the adenine H2 proton and the sugar H1' proton on the flanking base-pair in the 5'-direction (Weiss et al., 1984) has been used as a marker for minor groove

Non-exchangeable duocarmycin A protons in the complex The non-exchangeable duocarmycin A protons have been assigned by monitoring NOE and coupling connectivities along each of the two ring systems in the duocarmycin A–hairpin duplex complex. These chemical shift values are listed in Table 3 and establish that proton markers distributed throughout both rings of duocarmycin A can be monitored in the complex. Duocarmycin–duplex interactions We observe a large number of drug–DNA NOEs between duocarmycin and the hairpin duplex which are invaluable for defining the drug–DNA interactions associated with complex formation. These drug–DNA NOEs have been identified and some of these are shown as boxed crosspeaks in Figures 1A, 2A and 2B with assignments listed in the Figure captions. The complete list of drug–DNA NOEs in the duocarmycin A–hairpin duplex complex are listed in Table 3. The majority of the drug–DNA NOEs involve the inside concave edge A-subunit COOCH3-2, CH3-2, H4A,B, H4a, and H5A,B protons and B-subunit H3', H5' and OCH3-6' protons of duocarmycin and the minor groove protons on the DNA (Table 3). We outline below the drug–DNA NOEs involving the H3', H5' and OCH3-6' duocarmycin B-subunit protons and DNA protons that establish both the positioning of the concave edge of duocarmycin in the floor of the minor groove, as well as the directionality of the covalently bound drug in the complex.

Table 1 Exchangeable DNA proton chemical shifts (ppm) in the duocarmycin A–hairpin duplex complex and the chemical shift differences between the control hairpin duplex and the duocarmycin A–hairpin duplex complex at 15°C Base-pair C1·G14 C2·G13 T3·*A12+ T4·A11 T5·A10 T6·A9 C7·G8

T(NH3)

G(NH1)

A(H2)

Chemical shifts, ppm A(NH2-6)

12.22 (−2.00) 14.88 (+0.87) 14.05 (+0.11) 13.91 (+0.02)

8.03 (+0.67) 7.42 (+0.35) 7.67 (+0.60) 7.19 (−0.06) 12.61 (+0.08)

C(NH2-4) 8.21 (−0.07)a, 7.03 (−0.05)b 8.29 (−0.08)b, 7.01 (+0.05)b

13.08 (−0.00) 12.83 (−0.15) 8.98(NA)a 8.62(NA)b

8.27 (−0.00)b, 6.51 (−0.20)b

Sample was dissolved in 0.6 ml 90% H2 O/10% 2H2 O buffered solution containing 10 mM sodium phosphate, 0.1 mM EDTA (pH 7.0) at 15°C. The chemical shift differences between the control duplex and the duocarmycin A–hairpin duplex complex are listed in parentheses. A positive value in chemical shift difference indicates a downfield shift upon complex formation. a Hydrogen-bonded amino proton. b Exposed amino proton.

JMB—MS 450 Duocarmycin–DNA Complex

167

Figure 2. Expanded NOESY contour plots (250 ms mixing time) of the duocarmycin A–hairpin duplex complex in 2H2 O buffer (pH 7.0) at 25°C. A, The base proton (6.8 to 8.3 ppm) to the sugar H1' (5.1 to 6.4 ppm) region. The chain traces NOEs between the base protons and their own and 5'-flanking sugar H1' protons from C1 to C7, from G8 to G14, and from Ta to Te in the loop segment. The NOE crosspeaks A to R are assigned as follows: A: Duo(H5')T6(H1'); B: Duo(H5')-A11(H1'); C: Duo(H5')-A10(H1'); D: Duo(H3')T6(H1'); E: T6(H6)-C7(H5); F: A9(H2)-A10(H1'); G: A11(H2)A11(H1') and A11(H2)-A12(H1'); H: A11(H2)-T5(H1'); I: A10(H2)T6(H1'); J: A10(H2)-A11(H1'); K: A10(H2)-A10(H1'); L: A12(H2)A12(H1'); M: A12(H2)-G13(H1'); N: A12(H2)-T4(H1'); O: Ta(H6)-C1(H5); P: C1(H6)-C2(H5); Q: Duo(H3')A12(H1'); R: A9(H2)-A9(H1'). B, The base proton (6.9 to 8.4 ppm) to the 3.4 to 5.2 ppm region. The drug–DNA NOEs A to O are assigned as follows: A: Duo(H7)-G13(H4')/T6(H5'&H5"); B: Duo(H7)-T6(H5'&H5"); C: Duo(H7)-G13(H5'&H5"); D: Duo(H4B)-A11(H2); E: Duo(H4A)A11(H2); F: Duo(H5B)-A11(H2); G: Duo(H5B/H4a)-A11(H2); H: Duo(H4B)-A12(H2); I: Duo(H4A)A12(H2); J: Duo(H5B)-A12(H2); K: Duo(H5A)-A12(H2); L: Duo(H4a)A12(H2); M: Duo(6'-OCH3 )-A9(H2); N: A11(H2)-Duo(6'-OCH3 ); O: A10(H2)-Duo(6'-OCH3 ).

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Table 2 DNA proton and phosphorus chemical shifts (ppm) in the duocarmycin A–hairpin duplex complex at 25°C Base C1 C2 T3 T4 T5 T6 C7 G8 A9 A10 A11 A12 G13 G14 Ta Tb Tc Td Te

H8

H6

H5

7.65 7.58 7.66 7.43 7.36 6.93 7.17

5.77 5.59 1.73 1.69 1.60 1.50 5.09

7.76 8.12 8.06 7.99 8.18 7.52 7.45

H2

7.18 7.64 7.38 8.02 7.55 7.41 7.48 7.51 7.20

1.81 1.67 1.66 1.66 1.58

Chemical shifts, ppmb H1' H2' H2" H3'

H4'

H5'/H5"

5.83 6.03 6.27 5.90 6.07 5.20 5.59 5.59 5.77 6.08 5.35 5.32 5.60 5.82 6.11 6.07 5.96 6.04 5.91

4.21 4.20 4.33 3.95 2.40 1.65 3.83 4.18 4.38 4.49 3.20 4.04 3.47 4.27 4.22 4.14 4.00 4.16 4.06

4.06 4.10 4.11, 4.16 4.18 3.09, 3.64 3.47, 3.72 3.60, 3.92 3.56, 3.67 4.05, 4.16 4.20, 4.23 4.08, 4.13 2.43, 3.88 3.02, 3.94 3.95, 4.12 4.03, 4.14 3.92 3.81, 3.90 3.97, 4.06 NAa

2.21 2.13 2.35 2.01 2.03 1.50 2.20 2.43 2.69 2.65 2.19 2.70 2.36 2.40 2.21 2.20 2.05 2.06 2.03

2.40 2.45 2.72 2.38 2.29 1.99 2.20 2.76 2.86 2.86 2.19 2.65 2.57 2.55 2.51 2.43 2.28 2.32 2.33

4.82 4.83 4.93 4.67 4.59 4.37 4.46 4.79 5.03 5.07 4.71 4.85 4.85 4.80 4.76 4.76 4.67 4.64 4.80

31

Pc

−3.85 −4.04 −4.37 5.29 −4.70 −4.48 −4.26 −4.32 −4.37 −5.29 −5.05 −4.22

Sample was dissolved in 0.6 ml 2H2 O buffered solution containing 10 mM sodium phosphate, 0.1 mM EDTA, (pH 6.98) at 25°C. a NA, Not assigned due to severe overlap of the proton resonances. b Proton resonances that are upfield shifted relative to signals of other similar protons are underlined. c These are chemical shifts for the phosphorus atoms at (n)-P-(n + 1) steps with the value listed for residue n. Phosphorus resonances shifted outside the −4.0 to −4.5 ppm region are underlined.

The duocarmycin H5' proton (7.22 ppm) exhibits drug–DNA NOEs to the minor groove sugar H1' protons of T6, A11 and A10 (peaks A, B and C, respectively, Figure 2A) while the duocarmycin H3' proton (7.99 ppm) exhibits drug–DNA NOEs to the sugar H1' protons of T6 and A12 (peaks D and Q, respectively, Figure 2A) in the complex. Further, the duocarmycin H5' and H3' protons exhibit drug–DNA NOEs to the minor groove H2 protons of A10, A11 and the imino proton of T5 (peaks S and K, respectively, Figure 1A) in the complex. Similarly, the duocarmycin OCH3-6' proton

(4.26 ppm) exhibits NOEs to the imino protons of T5 and T6, as well as the minor groove H2 protons of A9, A10 and A11 (peaks M, O and N, Figure 2B) and the H1' protons of T6, A10 and A11 in the complex. These drug–DNA NOEs position the duocarmycin concave-edge H3', H5' and OCH3-6' protons on the B-subunit of duocarmycin in the minor groove centered about the (T4-T5T6)·(A9-A10-A11) segment. These results establish that the B-subunit is directed towards the 5'-direction relative to the *A12+ modification site on the hairpin duplex.

Table 3 Intermolecular NOEs between duocarmycin A and the nucleic acid protons in the duocarmycin A–hairpin duplex complex Drug protons Type NH-1 COOCH3-2 CH3-2 H4A H4B H4a H5A H5B H7 NH-1' H3' H5' OCH3-6' OCH3-7' OCH3-8' a

Shift (ppm) 4.41 3.90 1.90 3.81 3.62 4.99 4.98 4.62 8.26 11.12 7.99 7.22 4.26 3.96 4.11

vw, very weak; w, weak.

Nucleic acid protonsa T3(H1'), T4(H3') T4(H1'), T5(H3', H4', H5', H5") A11(H5', H5"), A12 (H2), G13 (H1', H2', H2", H3', H4', H5', H5"), G14(H1', H3', H4') T5(H1'), A10(H2)[vw], A11(H2), A12(H2, H1'), G13(H4')[vw] A10(H2)[vw], A11(H2), A12(H2, H1') T4(NH3)[vw],A10(H2), A11(H2), A12(H2) T4(NH3), T5(H1')[vw], A10(H2)[w], A11(H2), A12(H2), A12(H1')[vw] T4(NH3), A10(H2), A11(H2), A12(H2) T5(H4', H5', H5"), T6(H4', H5', H5"), A12 (H3')[vw], G13 (H4', H5', H5") T6(H1', H3')[vw], T6(H4', H5', H5"), A11(H1')[vw], A12(H4', H5', H5") T5(NH3), T6(H1'), A10(H2, H1'), A11(H2), A12(H1') T5(NH3), T6(H1', H4'), A9(H2), A10(H2, H1'), A11(H2, H1', H2', H2", H4') T5(NH3), T6(NH3, H1'), C7(H2', H2", H4'), A9(H2), A10(H2, H1'), A11(H2, H1', H3', H4') T4(H1'), C7(H5', H5"), A10(H1'), A11(H1', H4') C7(H4', H5', H5"), A10(H2), A12(H5', H5")

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Structure 5

Drug–DNA NOEs are also detected between the outside edge A-subunit NH-1 and H7 protons and B-subunit NH-1', OCH3-8' and OCH3-7' protons of duocarmycin and protons that line the minor groove walls of the DNA (Table 3). The A-subunit H7 proton (8.26 ppm) exhibits NOEs to the H5',5" protons of T6 and G13 (peaks A/B and C, respectively, Figure 2B) and the H4' proton of G13 (peak A, Figure 2B) in the complex. Similarly, the B-subunit NH-1' proton (11.12 ppm) exhibits NOEs to the H5',5" protons of T6 and A12 in the complex. The observed drug–DNA NOEs establish that both the A- and B-subunits of duocarmycin must penetrate into and be sandwiched between the walls of the minor groove since the duocarmycin outer edge protons exhibit NOEs to the sugar H5',5" protons that are part of the sugar-phosphate backbone of the DNA. Finally, the H4A,B and H4a duocarmycin protons that are involved in the two bonds linking the ring systems of the A-subunit of duocarmycin and the *A12+ residue on the hairpin duplex exhibit a large number of drug–DNA NOEs primarily to the minor groove H2 protons of A10, A11 and A12 in the complex (Table 3). These results confirm the covalent linkage of the duocarmycin to the minor groove N-3 atom of *A12+ on the hairpin duplex on complex formation and its positioning along the floor of the minor groove of the duplex. The large number of assigned drug–DNA NOEs listed in Table 3 should prove invaluable in determining the solution conformation of the duocarmycin–hairpin duplex complex.

phosphodiester backbones. The phosphorus resonances in the duplex segment of the complex have been assigned (Table 3) following analysis of a proton detected proton-phosphorus correlation experiment by linking individual phosphates to their 5'-flanking H3' protons and their 3'-flanking H4' and H5',5" protons in the complex. The upfield shifted phosphorus resonances include those at the A11-A12 (−5.29 ppm) and A12-G13 (−5.05 ppm) steps on the covalently modified strand and those at the T4-T5 (−5.29 ppm) and T5-T6 (−4.70 ppm) on the unmodified strand.

Phosphorus chemical shifts and assignments

The procedures for estimation of restraints for structure calculations on the duocarmycin–hairpin duplex complex are outlined in Experimental Procedures. These restraints can be summarized graphically to outline both the number and distribution at various sites on the complex (Figures 3(A)

Several phosphorus resonances in the duocarmycin–hairpin duplex complex in 2H2 O buffer (pH 7.0) at 25°C are shifted upfield of the −4.0 to −4.5 ppm range characteristic of unperturbed

Upfield shifted proton and phosphorus resonances in the complex An unusually large number of non-exchangeable sugar proton and phosphorus resonances shift upfield on formation of the duocarmycin–hairpin duplex complex. These upfield shifts are highlighted in Table 2 and depicted schematically in (Structure 5). These shifts are restricted to the A11-*A12+ -G13 segment on the modified strand and the T4-T5-T6 segment on the unmodified complementary strand in the complex. Note that there is a two base-pair offset between these upfield shifted resonances located on the modified and unmodified strands with the directionality of this offset similar to that observed for the shortest interstrand phosphorus–phosphorus distance across the minor groove. Distribution of restraints

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DEEP PAGE

Duocarmycin–DNA Complex

Figure 3. Distribution of restraints for individual nucleic acid residues and the drug molecule in the structure calculation of the duocarmycin–hairpin duplex complex. (A) The restraints have been classified as follows: non-exchangeable proton restraints (open rectangles) that were calculated on the basis of NOE buildups in 2H2 O; exchangeable proton restraints (hatched rectangles) that were calculated from the NOESY spectrum in H2 O and hydrogen bond restraints (filled rectangles) used to maintain base-pairing. Each intraresidue restraint was counted as one for that residue. Each interresidue restraint was counted as half for that residue and half for its partner in the interaction. The left panel lists the DNA–DNA restraints for individual residues and the right panel lists drug–drug restraints. (B) Distribution by residue position of intraresidue (open rectangles) and interresidue (filled rectangles) experimentally observed exchangeable proton and non-exchangeable proton restraints. The DNA–DNA NOEs are shown in the left panel and drug–drug NOEs in the right panel. (C) The drug–DNA NOEs involving specific DNA residues (left panel) and drug residues (right panel). Each drug–DNA restraint is counted as half for the DNA proton and half for its counterpart drug proton in the interaction. D, Pairwise r.m.s.d.s in the atomic coordinate positions calculated for each nucleic acid residue (left panel) and drug molecule (right panel) amongst the 4 relaxation matrix refined structures of the complex. The average values (open circles) are shown along with the extreme values (vertical bars).

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Duocarmycin–DNA Complex

Figure 4. A stereo view of the 4 superpositioned relaxation matrix refined structures of the duocarmycin–hairpin duplex complex. The duocarmycin is in yellow and the DNA duplex is in white with phosphate groups in red. The view is looking into the minor groove.

to (C)). The largest number of drug–nucleic acid restraints are centered about the (T4-T5-T6)·(A10A11-A12-G13) binding site for duocarmycin on the helical segment of the hairpin duplex (Figure 3C). The computations were undertaken on the complex consisting of the duocarmycin and the seven base-pair helical segment of the hairpin duplex. The molecular dynamics calculations were guided by 279 nucleic acid–nucleic acid restraints, 15 drug–drug restraints and 51 drug–nucleic acid restraints.

Structure calculations The NOE information was quantified and converted into distance restraints as described in Experimental Procedures and these were then used to calculate structures using the restrained molecular dynamics module in the XPLOR program. The two starting structures Init-A and Init-B ˚ ) were generated as described in (r.m.s.d. = 3.83 A Experimental Procedures. The protocols for the distance restrained molecular dynamics simulation are outlined in Experimental Procedures. The calculation yielded two distance refined structures ˚ for the seven base-pairs and with a r.m.s.d. of 1.00 A the drug molecule. These structures were next refined against the non-exchangeable proton NOE intensities while retaining the exchangeable proton distance restraints with the protocols for the relaxation matrix refinement outlined in Experimental Procedures. The dynamics were initiated with two different seeds for the random number generator which assigns initial velocities from a Maxwellian distribution to every atom on the system. The calculations yielded four intensity refined structures with an average pairwise r.m.s.d.

˚ for the same segment of the complex. of 0.83 to 1.05 A Stereo views of the four superpositioned relaxation matrix refined structures of the duocarmycin– hairpin duplex complex are shown in Figure 4. The distribution of pairwise r.m.s.d.s in the atomic coordinate positions calculated for each nucleic acid residue and drug molecule amongst the four relaxation matrix refined structures of the complex are outlined in Figure 3D. The relevant refinement statistics on the duocarmycin–hairpin duplex complex are summarized in Table 4. The quality of structures of the complex as judged by the R-factors and deviations from ideal covalent geometry are listed in Table 5. Structural features A view of the central (C2-T3-T4-T5-T6)·(A9-A10A11-*A12+-G13) segment looking normal to the helix axis and into the minor groove and another looking down the minor groove of one representative relaxation matrix refined structure of the duocarmycin–hairpin duplex complex are shown in Figure 5A and B, respectively. The covalently attached duocarmycin is positioned in the minor groove of the duplex such that its long axis is aligned with the minor groove axis and with the B-subunit directed in the 5'-direction relative to the *A12+ modification site (Figure 5A). The inner concave edge of the duocarmycin is positioned along the floor of the minor groove while the outer edge of the duocarmycin is bracketed by the walls of the minor groove such that the entire duocarmycin is positioned deep in the minor groove (Figure 5B). The corresponding space filling views of this segment of the complex are shown in Figure 6A and B, respectively.

JMB—MS 450 172

Duocarmycin–DNA Complex

Table 4 NMR refinement statistics for the duocarmycin A–hairpin duplex complex Starting structures Pairwise r.m.s.d. between the energy minimized Init-A and Init-B starting structuresa Distance refinement Number of distance restraints (includes 16 hydrogen-bonding restraints) ˚ ) for the 2 distance refined structures NOE violations (>0.2 A The r.m.s.d.s for the input distance restraints in the 2 distance refined structures Pairwise r.m.s.d.s between the 2 distance refined structuresa Relaxation matrix refinement Number of non-exchangeable proton intensity restraints (total of 4 mixing times) Number of distance restraints involving exchangeable protons ˚ ) for the 4 relaxation refined structuresb NOE violations (>0.2 A The r.m.s.d.s for the input distance restraints in the 4 relaxation refined structuresb Pairwise r.m.s.d.s amongst the 4 relaxation matrix refined structuresa

3.83 361 8–9 0.066–0.076 1.00 1016 61 2–4 0.300–0.314 0.83–1.05

a

The r.m.s.d.s calculated for the 7 base-pairs and the drug molecule. These NOE violations and r.m.s.d.s for input distance restraints are restricted to the 77 distance restraints (exchangeable protons including 16 hydrogen bonding restraints) that were used during relaxation matrix MD refinement. b

The stacking overlaps between the T3·*A12+ pair and its flanking C2·G13 and T4·A11 base-pairs in the complex are plotted in stereo in Figure 7A and B, respectively. For comparison the stacking patterns between the unmodified T4·A11 and T5·A10 base-pairs are plotted in stereo in Figure 7C. The stacking patterns of the A- and B-subunits of duocarmycin with the sugar phosphate backbone of the A11-*A12+ -G13-G14 segment on the modified strand and the sugar-phosphate backbone of the T4-T5-T6-C7 segment on the unmodified strand in the complex are plotted in stereo in Figure 8A and B, respectively.

Discussion Complex formation between duocarmycin A and the DNA hairpin (Structure 4) yielded a predominant species which was purified by HPLC. The purified complex gave well-resolved exchangeable and non-exchangeable proton spectra which have been analyzed by two-dimensional NMR approaches to obtain the base and sugar nucleic acid (Tables 1 and 2) and duocarmycin (Table 3) proton assignments. The NOEs between the duocarmycin

and the DNA have been identified and assigned in the complex (Table 3) and have yielded sufficient restraints to define the solution structures of the duocarmycin–hairpin duplex complex (Figure 5).

The covalent modification site Covalent modification between duocarmycin A and adenine 12 in the hairpin duplex results in protonation of this adenine in the adduct with charge delocalization over the aromatic ring system of the base (see Structure 3). This is confirmed by the downfield shifts of the H8 (8.18 ppm), H2 (8.02 ppm) and NH2-6 (8.98, 8.62 ppm) protons of *A12+ in the duocarmycin A–hairpin duplex complex (Tables 1 and 2). The weaker NOE between the imino proton of T3 and the H2 proton of *A12+ and the 2.0 ppm upfield shift of the imino proton of T3 on complex formation (Table 1) establishes a weaker Watson-Crick pairing for the T3·*A12+ pair in the duocarmycin–hairpin duplex complex. The structure calculations were undertaken with only a single T3(N1H) to *A12+ (N3 ) ˚) hydrogen bonding restraint defined by lower (2.4 A

Table 5 Quality of structures for the duocarmycin–hairpin duplex complex Parameter

Initial structures

The r.m.s. deviations from ideal covalent geometry ˚) Bond length (A 0.008, 0.008 Bond angles (°) 3.50, 3.47 Impropers (°) 0.331, 0.353 R-factorsa Unweighted R1 0.800, 0.401 Weighted R1 1.648, 1.300 R1/6 0.176, 0.089 Convergence The r.m.s.d.s relative to ˚ )b averaged structure (A NA a

Distance restrained MD structures

Relaxation matrix MD structures

0.011, 0.011 3.88, 3.92 0.315, 0.312

0.009–0.010 3.07–3.78 0.310–0.321

0.375, 0.372 0.313, 0.336 0.071, 0.071

0.176–0.182 0.270–0.292 0.042–0.045

NA

0.56–0.68

The definitions for the various R-factors are taken from Nilges et al., (1992). b The r.m.s.d.s are relative to the average of the 4 relaxation matrix refined structures of the complex containing all 7 base-pairs and the drug molecule.

JMB—MS 450 Duocarmycin–DNA Complex

173

Figure 5. Two views of a representative relaxation matrix refined structure of the duocarmycin–hairpin duplex complex. The duocarmycin is in yellow while the d(C2-T3-T4-T5-T6)·d(A9-A10-A11-*A12+-G13) DNA segment is in magenta with the backbone highlighted in blue. (A) View looking into the minor groove and normal to the helix axis. The A and B-rings of duocarmycin are labeled as are the nucleic acid residues. Duocarmycin forms a covalent linkage to N-3 of A12 in the complex. (B) View looking down the minor groove establishing bracketing of the entire duocarmycin within the walls of the minor groove. The duocarmycin B ring projects towards the viewer while the A ring projects away from the viewer in this view.

˚ ) bounds. This distance adopts a and upper (2.8 A ˚ in the refined structure of the value of 2.31 (20.03) A duocarmycin–hairpin duplex complex consistent with the T3·*A12+ pair aligning through weak hydrogen bond formation. This may be a consequence of positive charge delocalization to N-3 of adenine on adduct formation which repels an otherwise thymine imino proton donor involved in hydrogen bond formation. DNA conformation in the complex All the non-modified base-pairs retain standard Watson-Crick alignments in the duocarmycin–hairpin duplex complex. The x-displacement values of

the base-pairs from the helix axis are centered about ˚ which is characteristic of a B-DNA helix for −0.5 A the complex. The glycosidic torsion angles for all non-modified bases are in the anti range while the majority of the sugars adopt pseudorotation angles in the P = 126° (C-1'-exo) to P = 162° (C-2'-endo) range. The only exceptions are C7 (P = 60°) which is a terminal residue and its neighbor T6 (P = 96°). Both the helical rise and helical twist values are characteristic of a minimally perturbed righthanded helix in the complex. The propeller twists for the T·A pairs in the d(T3-T4-T5-T6)·d(A9-A10-A11-*A12+ ) segment of the complex are largest for the central T4·A11 (−31°) and T5·A10 (−29°) pairs and smaller for the

JMB—MS 450 174 flanking T3·*A12+ (−14°) and T6·A9 (−3°) pairs. Large propeller twists for A·T base-pairs of the order of 25° have been previously observed for the central segment of an A6·T6 tract in the crystal structure of a DNA oligomer duplex (Nelson et al., 1987). The base-pair stacking at the three steps centered about the adduct site in the duocarmycin–hairpin duplex complex are shown in Figure 7. The stacking between the T3·*A12+ and T4·A11 base-pairs (Figure 7B) is similar to that between the T4·A11 and T5·A10 base-pairs (Figure 7C) with intrastrand stacking observed between the purines and between the pyrimidines on individual strands. A similar stacking pattern has been observed between adjacent A·T base-pairs for an A6·T6 tract in the crystal structure of a DNA oligomer duplex (Nelson et al., 1987). By contrast, the overlap between the

Duocarmycin–DNA Complex

C2·G13 and T3·*A12+ base-pairs (Figure 7A) results in stacking between the purines but not the pyrimidines on individual strands of the complex. Duocarmycin alignment in the minor groove Two single bonds covalently link the N 3 position of *A12+ and ring A of the drug in the duocarmycin A–hairpin duplex complex. The corresponding torsion angles are 129.2° (A12[C4 ]-A12[N3 ]-Duo[C4 ]Duo[C4a ]) and −77.7° (A12[N3 ]-Duo[C4 ]-Duo[C4a ]Duo[C5 ]) in the refined solution structure of the complex. These torsion angle values orient the covalently attached duocarmycin A in the 5'-direction relative to its modification site. The covalently bound duocarmycin adopts an extended conformation in the minor groove with its long axis aligned 045° relative to the helix axis

Figure 6. Corey-Pauling-Koltum space filling views of the central segment of a representative relaxation matrix refined structure of the duocarmycin–hairpin duplex complex. The duocarmycin is shown in yellow and the DNA is shown in white except for the phosphates, which are shown in red. (A) View looking into the minor groove. (B) View looking down the minor groove.

JMB—MS 450 175

Duocarmycin–DNA Complex

Figure 7. Stereo views of base-pair stacking in a representative relaxation matrix refined structure of the duocarmycin–hairpin duplex complex. The C2 ·G13, T3·*A12+, T4·A11 and T5·A10 base-pairs in the complex are colored magenta, blue, red and green, respectively. The duocarmycin covalently linked to the N 3 position of A12 is colored yellow. Stacking overlaps (A) between C2·G13 and T3·*A12+ pairs, (B) between T3·*A12+ and T4·A11 pairs and (C) between T4·A11 and T5·A10 pairs.

(Figure 5A). The duocarmycin A ring and its substituents span the d(C2-T3-T4)·d(A11-*A12+ G13) segment centered about the *A12+ covalent modification site while the B-ring spans the d(T4-T5-T6)·d(A9-A10-A11) segment (Figure 5A). Duocarmycin is positioned deep in the groove with the non-polar concave edges of both subunits directed towards the floor of the minor groove while the polar edges are directed outwards towards solvent (Figure 5B). The covalently bound duocarmycin A tracks the walls of the minor groove and consequently there is a 25.2° twist relating the A-ring and the B-ring in the structure of the duocarmycin– hairpin duplex complex (Figure 5B). The positioning of the A and B-rings of duocarmycin A within the walls of the d(T4-T5-T6C7)·d(A11-A12-G13-G14) segment is best depicted by the overlap patterns of the extended duocarmycin A with the backbone of the modified strand in Figure 8A and the unmodified strand in Figure 8B. This overlap geometry should result in upfield shifts of the protons and phosphorus atoms in the sugarphosphate backbone of this segment in the complex. The overlap geometries (Figure 8A and B) are

consistent with the results summarized in Structure 5 with upfield shifts detected at the minor groove protons (H1' and H4'), backbone protons (H5',5") and phosphorus atoms of the d(A11-*A12+ -G13) segment on the modified strand and the d(T4-T5-T6) segment on the unmodified strand in the complex. Minor groove width at the drug-binding site Duocarmycin binds to the d(T3-T4-T5-T6)·d(A9A10-A11-*A12+ ) segment of the hairpin duplex and the dimensions of the minor groove spanning the binding site are of interest. We have measured the width of the minor groove by monitoring the shortest phosphorus-phosphorus separation across this groove in the refined solution structures of the duocarmycin–hairpin duplex complex. The average ˚ separations across the minor groove are 11.0 A ˚ (T5pT6 to *A12+pG13) (T4pT5 to G13pG14), 9.0 A ˚ (T6pC7 to A11p*A12+ ) for this segment and 10.8 A of the complex. These values for the width of the minor groove centered about the bound duocarmycin in the complex are similar to the minor ˚ ) and groove width in B-form DNA (10.0 to 12.0 A

JMB—MS 450 176 much narrower than the corresponding value in ˚ ). A-form DNA (17.0 A Hydrophobic drug–DNA interactions A large set of drug–DNA NOEs have been identified between the inwardly facing hydrophobic concave edge of duocarmycin A and the adenine H2 and sugar H1' protons that line the floor of the minor groove of the d(T3-T4-T5-T6)·d(A9-A10-A11*A12+ ) segment of the DNA (Table 3). The solution structure of the complex based on the magnitude of these drug–DNA NOEs establishes that there is no room for water molecules to be sandwiched between the duocarmycin A and the minor groove floor of the DNA. Further, drug–DNA NOEs have also been identified between the hydrophilic outwardly facing edge of duocarmycin and the sugar H4' and H5',5" protons of the sugar-phosphate backbone that line the walls of the minor groove of the DNA (Table 3). Indeed, duocarmycin A is enveloped by the walls of the minor groove of the DNA at its binding site in the complex. The only hydrogen bonding interaction detected between the drug and the DNA involves the 8-OH hydroxyl proton on ring A of duocarmycin A and the phosphate oxygen at the G13pG14 step on the modified strand in the complex. This distance ˚ in the exhibits an average value of 2.13 (20.05) A refined structures of the complex.

Duocarmycin–DNA Complex

Comparison with other covalent drug–DNA complexes The covalent duocarmycin A-adenine adducts are most closely related to the covalent CC-1065-adenine adducts for which extensive chemical, physical and biological experiments have been reported previously (Hurley et al., 1988; Boger et al., 1991b). CC-1065 (2) binds covalently to the N 3 position of adenine and exhibits a sequence specificity (Reynolds et al., 1985) similar to that found for duocarmycin A (Boger et al., 1990). Structural studies have established that the CC-1065-adenine adduct at the duplex level is primarily stabilized through drug–minor groove van der Waals interactions (Scahill et al., 1990; Powers & Gorenstein, 1990; Lin & Hurley, 1992). Related structural studies have also elucidated the charge delocalization over the protonated adenine ring of the adduct (Lin & Hurley, 1990) and its hydration environment (Lin et al., 1991). Duocarmycin (Structure 1) contains one less ring than CC-1065 (Structure 2) and the solution structure of the duocarmycin–hairpin duplex complex reported in this study should provide structural insights into common motifs utilized by the family of drugs that bind to DNA through alkylation at the N3 position of adenine. In general, these drugs adopt extended conformations, are positioned centrally in the narrow minor groove at A·T-rich sites and exhibit extensive hydrophobic interactions between the drug and both the floor and the walls of the minor groove.

Figure 8. Stereo views normal to the average plane of the A and B rings of duocarmycin in a representative relaxation matrix refined structure of the duocarmycin–hairpin duplex complex. (A) Stacking of the A11-*A12+ -G13-G14 sugar-phosphate backbone over duocarmycin. (B) Stacking of the T4-T5-T6-C7 sugar-phosphate backbone under duocarmycin.

JMB—MS 450 177

Duocarmycin–DNA Complex

The CC-1065-DNA and duocarmycin–DNA complexes which involve alkylation at the minor groove N 3 position of adenine exhibit structural features that are distinct from covalent anthramycin–DNA (Krugh et al., 1989; Boyd et al., 1990), mitomycin– DNA (Sastry et al., 1995) and benzo[a]pyrene-DNA (Cosman et al., 1992; De los Santos et al., 1992) complexes which involve alkylation at the minor groove N 2 exocyclic amino position of guanine. The former N 3-adenine covalent adducts are completely embedded within the walls of the narrow minor groove of the duplex while the latter N 2-guanine covalent adducts involve chromophores that are asymmetrically positioned in a widened minor groove with one face of the aromatic chromophore stacked over the partner strand and the other face exposed to solvent. The coordinates of the duocarmycin A–hairpin duplex complex have been deposited with the Protein Data Bank, Brookhaven National Laboratory, Upton, New York, 11923 U.S.A. (Accession number: 107D).

Experimental Procedures Sample preparation Duocarmycin A (Structure 1) was obtained from Dr Hiromitsu Saito of the Kyowa Hakko Kogyo Company, Japan and used without further purification. The 19-mer DNA–hairpin sequence (Structure 4) was synthesized on a 10 mmol scale using synthesis and purification procedures reported previously (Sastry & Patel, 1993). The duocarmycin A–hairpin duplex complex was prepared by adding two equivalents of duocarmycin A to 19-mer DNA hairpin sequence (11 mM) in 5 ml of a 200 mM NaCl, 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.0) buffered solution containing 20% (v/v) methanol. The pH of the solution was adjusted to 7.0 and the reaction mixture was stirred in the dark at 4°C. The major adduct (>80% of product) was purified by two successive semi-preparative HPLC treatments on a C18 ODS Hypersil column (Keystone Co.) using a 20 mM sodium phosphate/ methanol gradient over a period of 90 minutes. Three minor adducts (<20% of product) were resolved on HPLC but were not characterized further. The HPLC purified drug–hairpin complex was then dialyzed in 1 mM sodium phosphate buffer (pH 7.0) at 4°C for six to eight hours. The desalted adduct was then lyophilized to dryness and dissolved in 0.6 ml of 10 mM sodium phosphate, 0.1 mM EDTA buffer. The final concentration of this NMR sample is 3.2 mM. NMR experiments One and two-dimensional NMR data on the duocarmycin–hairpin duplex complex used acquisition and processing procedures reported previously (Sastry & Patel, 1993). Proton NOESY spectra on the complex in 2H2 O solution at 25°C were collected both for assignment (mixing times 50, 150 and 250 ms; relaxation delay 2.0 seconds) and NOE buildup (mixing times 40, 80, 120 and 160 ms; relaxation delay 5.0 seconds) purposes. Proton NOESY data sets on the complex were recorded at mixing times of 120 and 170 ms in H2 O solution at 15°C using the

jump and return read pulse sequence (Plateau & Gueron, 1982) with a 1.5 seconds repetition delay. The phase-sensitive COSY and HOHAHA (spin lock times of 50 and 75 ms) spectra on the complex in 2H2 O buffer at 25°C were obtained with a repetition delay of 1.5 second. A heteronuclear proton-detected 1H-31P COSY experiment (Sklenar et al., 1986) on the complex was recorded at 25°C. Structure calculations The interproton distance restraints for the duocarmycin– hairpin duplex complex were obtained from the NOE buildup experiments (40, 80, 120, and 160 ms mixing time) in 2H2 O buffer at 25°C and from a 120 ms mixing time H2 O NOESY data set recorded at 15°C. The distances involving the base–base, base–sugar and the drug–DNA non-exchangeable protons in the complex were calculated using ˚ as the reference. The the cytidine H5–H6 distance of 2.45 A distances involving methyl groups were calculated using ˚ as the reference. The the thymine H5–CH3 distance of 2.9 A distances involving the intraresidue sugar to sugar protons ˚ were calculated using the sugar H2'–H2" distance of 1.8 A as the reference. In general, the distance bounds were ˚ for the non-overlapping non-exchangeable set at 20.5 A ˚ for the methyl groups and proton proton pairs and 20.8 A pairs involving exchangeable protons. Duocarmycin A, as well as the A and B-forms of the d(C-C-T-T-T-T-C)·d(G-A-A-A-A-G-G) duplex (without the T5 loop) were generated using the INSIGHT II program (Biosym). Two initial starting models of the duocarmycin A–d(C-C-T-T-T-T-C)·(G-A-A-A-A-G-G) complex (designated Init-A and Init-B) were constructed by docking the duocarmycin A molecule onto the A-form and the B-form DNA duplexes in a manner that qualitatively attempted to satisfy the NOE-based distance restraints. The molecular dynamics simulations were carried out on a Silicon Graphics Challenge Server using the XPLOR program (A. Brunger, Yale University) in which all hydrogen atoms are treated explicitly. The SHAKE algorithm was used to constrain bond lengths involving hydrogen atoms during the molecular dynamics simulation. Each of the initial structures was subjected to 500 cycles of energy minimization (without NOE restraints) to relieve bad contacts between non-bonded atoms. In the course of energy minimization, hydrogen bond restraints for non-modified DNA base-pairs were used to retain the Watson-Crick alignment along the DNA duplex. Distance restrained molecular dynamics calculations All the molecular dynamics calculations were carried out on SGI Indigo or Crimson work stations using the XPLOR program (A. Brunger, Yale University) using procedures described previously (Sastry et al., 1995). The molecular dynamics calculations were performed in vacuum with reduced phosphate charges and a distancedependent dielectric was used to account for the screening effect of solvent and counterions. Distance restrained molecular dynamics calculations with initial velocities assigned from a Maxwellian distribution at 1000 K were performed for a total of 20 ps on each of the two energy minimized Init-A and Init-B starting structures. The restraints were introduced gradually over 20 ps (0.25 fs time step) with the weights ˚ 2 for the non-exincreased from 1.0 to 32.0 kcal/mol A changeable proton restraints, increased from 1.0 to ˚ 2 for the exchangeable proton restraints and 8.0 kcal/mol A ˚ 2 for the hydrogen increased from 1.0 to 60 kcal/mol A

JMB—MS 450 178 bond restraints. The system was next cooled gradually to 300 K over 4.2 ps (0.5 fs time step) with retention of the full scale of distance restraints. The system was subjected to an additional 12 ps of restrained molecular dynamics at 300 K. The coordinates were averaged over the last 5 ps and subjected to 500 steps of conjugate gradient minimization to give the distance refined DR-A and DR-B structures.

Relaxation matrix refinement The two distance refined structures were refined against the NOE crosspeak intensities with the relaxation matrix (relax) refinement routine of the XPLOR program. The NOE intensities (total 1016 based on four NOESY data sets collected at mixing times of 40, 80, 120 and 160 ms) were incorporated as penalty functions in the relax energy term, in which exponent 1/6 was used. We also included NOEs that were in overlapped regions at this stage of the refinement. An isotropic correlation time of 2.75 ns derived from a systematic grid search, along with a cutoff distance ˚ was used during the relaxation matrix refinement of 5.5 A calculations. The distance refined structures were subjected to 1 ps of molecular dynamics at 1000 K during which the weights for the non-exchangeable NOE intensities were increased from 5 to 400 kcal/mol while weights for the non-exchangeable distance restraints were ˚ 2. The weights for the reduced from 32 to 0 kcal/mol A hydrogen bonding distances and distance restraints involving exchangeable protons were retained and were the same as used in the distance restrained molecular dynamics refinement. The system was next gradually cooled to 300 K over 2.25 ps (0.5 fs time step) with retention of the full scale of restraints. This was followed by 4 ps of molecular dynamics (time step 1 fs) at 300 K. The coordinates during the last 1.0 ps of dynamics were averaged and these averaged coordinates were subjected to energy minimization. The calculations were repeated with a different seed for the random number generator yielding a total of four relaxation matrix refined structures. An averaged structure, with mean positions for all atoms was computed for the four relaxation matrix refined structures of the complex. This average structure, which was not energy minimized, provides a reference for evaluating the quality and convergence statistics amongst the four relaxation refined structures of the duocarmycin A–hairpin duplex complex.

Structure analysis Helical and sugar-phosphate backbone parameters were calculated on the duocarmycin A–hairpin duplex complex using the CURVES program (Lavery & Sklenar, 1988). The structures were displayed, analyzed and plotted using Insight II (Biosym Technologies, Inc.).

Acknowledgements This research was supported by NIH grant CA46778 to D.J.P. We thank Dr Hiromitsu Saito of the Kyowa Hakko Kogyo Company, Japan for a generous sample of duocarmycin A.

Duocarmycin–DNA Complex

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JMB—MS 450 Duocarmycin–DNA Complex

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Edited by P. E. Wright (Received 30 November 1994; accepted 30 January 1995)