Eur. J. Biochem. 203, 505-511 (1992) cc? FbBS 1992

Three-dimensional solution structure of apo-neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy elisabeth ADJADJ, Eric QUINIOU, Joel MISPELTER, Vincent FAVAUDON and Jean-Marc LHOSTE

U 219 INSERM, lnstitut Curie, Biologie, Centre universitaire, Orsay, France (Received July 26/September 13, 1991) - EJB 91 0996

The three-dimensional solution structure of apo-neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined p-sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of aponeocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven-stranded antiparallel /?-barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo-protein complex and for the chromophore specificity within the antitumoral family.

Neocarzinostatin, isolated from the culture broth of Streptomyces carzinostaticus var. F41 (ATCC 15944) [ 11, belongs to a large protein antibiotic family including actinoxanthin, auromomycin, mitomalcin, lymphomycin and actinocarcin. It is a complex consisting of a fluorescent chromophore tightly but not covalently bound 121 to a 10.7kDa protein containing 113 amino acid residues [3]. This chromophore, the very unusual molecular structure of which is shown in Fig. 1 [4-61, exhibits the full biological activity of neocarzinostatin [7]. It binds to DNA with high affinity and produces DNA damage by radical reactions [8 - 111. The protein component termed apo-neocarzinostatin has no cytotoxic activity: it primarily serves as a carrier protecting the chromophore from decomposition (hydrolysis) [12] and can be internalized in normal or virus-transformed eukaryotic cells 113, 141. Actinoxanthin and auromomycin have similar functions but with different specificity regarding nature and activity of the chromophore [15- 171. Despite these specificities, the apo-proteins of neocarzinostatin, auromomycin and actinoxanthin are similar in size and have about 45% sequence similar (Fig. 2). The crystal structure of these proteins has been reported at different resolution: 0.16 nm for the apoprotein of auromomycin currently termed macromomycin (MCR) [17], 0.25 nm for actinoxanthin (AXN) [16] and 0.35 nm for neocarzinostatin (NCS) [18]. Each structure comprises

a seven-stranded antiparallel /?-barrel which forms, with two antiparallel /?-ribbons, a characteristic cavity, the probable binding site for the chromophore in the active complex [16181. Since the overall size and global folding of the binding site is similar in the three proteins, the chromophore specificity may arise from properties of residues which interact directly with the ligand [17]. It is therefore of interest to compare the sequence of residues involved at the surface of the binding hole of these proteins. Sequential assignment and secondary structure of aponeocarzinostatin have been recently elucidated by 'H NMR [19, 201. Now, a set of distance constraints derived from the NOESY maps has been utilized in calculations of three-dimen-

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Correspondence to J.-M. Lhoste, U219 INSERM, Institut Curie, Biologie, Centre universitaire, B i t 112, F-91405 Orsay cedex, France. Ahhreviutions. 2D, two-dimensional; COSY, 2D scalar correlated spectroscopy; NOESY, 2D nuclear Overhauser enhancement spectroscopy; rms, root mean square.

HO

NWh

H

Fig. 1. Chemical structure of neocarzinostatin-chromophore.

506 10

1

20

30

40

M CR

-APCVTVTPATGLSNGOTVTVSATGLTPGTVYHVGOCAVV

NC S

AAPTATVTPSSGLSDGTVVKVAGAGLQAGTAYDVGQCAWV

AXN

-APAFSVSPASGLSDGQSVSVSGAAA-GET-YYIAQCAPV

MCR

EPGVIGCDATTSTDVTADAAGKITAQLKVHSSFQAVVGAD

NCS

DTGVLACDPADFSSVTADANGSASTSLTVRRSFEGFLF-D

AXN

G-GQDACNPATATSFTTDASGAASFSFTVRKSYAGQTP-S

II

I I

itti

I

III 1 - 1

1 I

I

IIIII

I I I I

I I

Ill I

70

I

I I

II

I II I I l l I

too

90

80

iiiii I

60

It

II II

II I

I I I t

I I I I I I

50

II

I I

I

I

110

M CR

GTPWGTVNCKVVSCSAGLGSDSGEGAA-QAITFA

NCS

GTRWGTVDCTTAACQVGLSDAAGNGPEGVAISFN

AXN

GTPVGSVDCATDACNLGAGNSGLNLG-HVALTFG

I I Ill1 I

I I

I

I Ill I II

II

I I

I

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II I

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Fig. 2. Comparison of the amino acid sequences of apo-neocarzinostatin (NCS) [19], macromomycin (MCR) and actinoxanthin (AXN) 131. The sequence numbering system is that of apo-neocarzinostatin.This sequence alignment was done with respect to the 8-strand regions of each protein structure.

sional structures of apo-neocarzinostatin. As previously suggested from preliminary data [19, 201 and in agreement with the partially refined X-ray structure [18], the solution structure of apo-neocarzinostatin has been found to be closely similar to that of macromomycin and actinoxanthin and presents a deep cleft, thought to be the binding site. EXPERIMENTAL PROCEDURES Protocol for the protein structure determination

constant, T is the absolute temperature, S is a scale factor, and A: and 0, are the positive and negative error estimate of r i j , respectively. The program allows an adjustment of the force constant through the scale factor S. This was done in three steps starting with S = 1 to a final value of S = 50 to reach a force constant equal to 2500 kJ mol-’ nm-2. In addition, the nonbonded interactions were switched off using a cutoff of 1 nm. This procedure greatly reduced the computation time, but the list of nonbonded interaction should be updated every 20 iterations. Finally, 10 of the 25 refined structures with the lowest total and lowest NOE constraint energies were subjected to restrained molecular dynamics to explore the conformational space by simulated annealing. At this stage, the sequential NH-CaH NOE constraints as well as the hydrogen bonding informations were removed [29]. Thus, the molecular dynamic calculations were performed using solely the 91 6 inter-residue distance restraints which were described by the same potential energy used in the molecular energy minimization but keeping the force constant at its maximum value of 2500 kJ mol-’ nm-2. The protocol was essentially identical to that described in the literature [29, 301. It comprises a 1-ps heating period to 1200 K followed by a 4-ps period of equilibration at 1200 K with a temperature relaxation time T of 0.2 ps [31]. Then, the structure was cooled over a period of 6 ps with z = 2 ps to a target temperature of 0 K. Finally, the refinement was achieved after 1000 steps of conjugate gradient minimization. All molecular calculations were performed on a Silicon Graphics Iris 4D/210 C T X B using as mentioned above the INSIGHT/DISCOVER software package. However, analysis of the results was done using home-written programs running on a Compaq 386/33 personal computer. NMR constraints

Most of the restraints used in the calculations described above were obtained from NOESY spectra. This requires an assignment of the NOESY cross-peak intensities to interproton distances. But, due to severe overlap of resonances, even at 600 MHz, only 724 cross-peaks could be initially assigned without ambiguity. However, using a similar structure [25] allowed us to enhance the constraint number to 916 and to assign correctly hydrogen bonding to 30 NH-CO couples of the protein. In fact, the observation of a slowly exchanging amide proton only indicates that it is implicated in an hydrogen bond, but gives no information with respect to the oxygen with which it interacts, except in the well defined p-sheet regions of the protein. Together with 88 sequential NH-CaH distance constraints, this led to the 1034 constraints used in the structure computation. This approach using a possible structure is justified here by the fact that the initial analysis of secondary structure elements and folding of neocarzinostatin [19] has indicated that its structure should be very similar to that of macromomycin [17]. Thus, using the sequence alignment proposed in Fig. 2 and the HOMOLOGY package (Biosym Technologies Inc., San Diego, California), a structure was obtained which was energetically relaxed by restrained energy minimization using the unambiguous 724 inter-residue restraints. Then, a theoretical NOESY spectrum was generated from the full relaxation matrix [32] corresponding to that structure with an isotropic overall correlation time (7,) of 3 ns. It confirmed the where r i j and r$ are the calculated and target distances, respec- correctness of the assignment and, further, it removed some tively, and C1 and C2 are constants given by C1 = 0.5 kBTS ambiguities on additional cross-peaks due to resonance over( A $ ) - 2 and C2 = 0.5 k B T S ( A ; ) - ’ , where k , is the Boltzmann lap.

The final set of structures described below has been obtained in three main steps as follows. First, 45 structures were generated, using DSPACE (Hare Research Inc., Woodinville, Washington) based on the distance geometry algorithm [22, 231, from a total of 1034 NMR constraints including 30 hydrogen bond constraints derived from the exchange properties of amide protons [19], 88 sequential NHCclH distance restraints and 91 6 other distance constraints. After optimization of these structures using a penalty function, two families of 20 and 25 structures, respectively, were obtained as ‘topological mirror images’ [24]. The choice between these two families was made using a reference structure of neocarzinostatin obtained by homology [25] from the known X-ray crystal structure of macromomycin [17] (see below). Twenty topologically incorrect structures were therefore rejected for the second step of the computation. The remaining 25 structures were then subjected to a multi-step restrained energy minimization using the ‘Consistent Valence Force Field’ [26] from the INSICHT/DISCOVER software package (Biosym Technologies Inc., San Diego, California). The interproton distance constraints were taken into account by an extra potential energy term in the form of a skewed biharmonic effective potential given by [28]:

507 Table 1. Average rms deviations from average structure for the 10 best refined structures. Only heavy atoms are considered. The /j'-sheet residuesare:4-6,17- - 24,30- 36,53 - 57,63 - 69,93 -96 and 108111. Amino acids

Structure

4-113

backbone side chain all heavy backbone sidc chain all heavy

8-Shect residues

Distance (rmsd) nm 0.176 k 0.032 0.221 k 0.031 0.199 k 0.030 0.058 f 0.006 0.156 f 0.015 0.085 _+ 0.007

Table 2. Summary of residual constraint violations. Range distance nm 0.01 -0.02 0.02-0.03 0.03 -0.04 0.04-0.05

Average number of distance constraint violation

23.9 4.5 1.2 0.1

All the distance restraints were then classified into three ranges corresponding to the intensities of the assigned crosspeaks calibrated from 50-ms and 200-ms mixing-time NOESY spectra, keeping in mind the problem of spin diffusion. In this respect, calculation of the full relaxation matrix [32] at different stages of the structure computation allowed one to determine more accurate intensities. The upper bounds used as constraints were 0.25 nm, 0.4 nm and 0.5 nm corresponding to strong, medium and weak intensity of the NOE cross-peaks, respectively, while the lower bounds were always taken to be the sum of the van der Waals radii.

RESULTS The statistics among the 10 final structures are summarized in Table 1. The overall atomic rms difference with the coordinates of the average structure is of0.176 _+ 0.032 nm for the backbone atoms and 0.199 0.030 nin for all heavy atoms excluding the N-terminal residues, Alal to Pro3, which are very poorly defined. All these structures satisfy the experimental data and none exhibits a distance constraint violation greater than 0.05 nm (Table 2). The backbone conformation is shown in Fig. 3 where all 10 structures are superimposed. With the exception of Tyr32 and Ah59 which have a positive average value of 4, the backbone torsion angles for all non-glycine residues fit the allowed region of the Rainachandran plot (Fig. 4). Clearly, all the regions comprising the elements of well defined secondary structures exhibit a good superimposition (Fig. 3), resulting in an average rms deviation from the mean structure of 0.058 nm for the backbone atoms (Table I and Fig. 5). In contrast, the regions comprising respectively residues 41 43, 49 - 52, 78 - 82, 99 - 106 exhibit an average atomic rms deviation larger than 0.2 nm for the backbone (Fig. 5). They are implicated in loops for which NOESY data are sparse probably due to their inherent flexibility.

Description of the structure

Closely similar to the structures of actinoxanthin [16] and macromomycin [17], the structure of neocarzinostatin consists of a flattened seven-stranded antiparallel /3-barrel arranged in a Greek key [33] and two short external loops which lie at the base of the barrel somewhat perpendicular to one another (Fig. 6). One of the two loops is closed by the Cys37 - Cys47 disulfide bridge and the other one runs from residues Ser72 to Asp87. The barrel and the two loops build up a deep cavity, the active site. The barrel is composed of two layers of antiparallel 8sheets (Fig. 6). The external layer is formed by strands 1 , 2 and 3 containing residues 4 - 6, 3 7 - 24 and 63 - 69, respectively. Strand 2 is hydrogen-bonded to strand 3 (Table 3) and both form a well defined /3-sheet. The flexibility of strand 1, which contains the N-terminal, precludes an accurate determination of the hydrogen-bonding interaction with strand 2. These two last strands are connected by a large loop extending from Val7 to Gly16 and beginning with two unusual turns at residues Va17-Thr8 and Pro9 - Serll. The NH and the 01' of Thr8 are hydrogen-bonded to the CO and the NH of Lys20 (Table 3), respectively. It is interesting to note (Figs 3 and 5) that the second part of this loop (from Serll to Gly36) is relatively well defined. This suggests a greater rigidity for this pcptide segment. At this point, it must be mentioned here that the relative position of strands 1 and 2 proposed previously on the basis of a dNN(6,21) connectivity [19], must be corrected. In fact, CaH-CaH cross-peaks between Ala5 and Gly23 have been now identified in the NOESY map. The correct positioning of strand 1 with respect to strand 2 requires a two-residue shift as compared to the folding already described [I 91. The internal layer of the barrel comprises strands 4, 5 , 6 and 7 which include residues 53 - 57, 30 - 36, 93 - 96 and 108 - 111, respectively. Val69 CO has a bifurcated hydrogen bond to the NHs of Glyl6 and Thrl7 in eight structures (Table 3). Strand 5 is hydrogen-bonded to strand 4 on one side and to strand 6 on the other side. The hydrogen-bonding pattern between strand 6 and the C-terminal containing strand 7 is very reduced. Only one hydrogen bond between Cys93 CO and llellO N H could be characterized in all 10 structures, consistent with its slow exchange rate [19]. Another hydrogen bond involving Val108 NH and Val95 CO could be observed in only four structures. Finally, a hydrogen-bonding interaction between Leu13 NH and S e r l l l CO helps to maintain the spatial proximity between the C-terminus and the loop connecting strand 1 and strand 2. At the opposite side, the barrel is bounded by three unusual loops which link, respectively, strands 2 and 5 (residues 25 29), strands 3 and 4 (residues 58-61) and strands 6 and 7 (residues 97-107). All these loops are flexible as suggested by a lack of NOESY connectivities (Fig. 5). As a consequence, the calculated structures exhibit large deviations for these regions. Especially, the loop between strands 6 and 7 involved in the active site could adopt a different conformation when the chromophore is bound. However, a hydrogen bond between Leu26 NH and Gly61 CO and hydrophobic interactions between Leu26, Tyr32, Leu97 and Pro105 could play a key role in stabilizing this region of the protein [34]. The other important unit of the neocarzinostatin structure contains the two external twisted antiparallel p-ribbons formed by residues 37 - 47 and 72 - 87 (for the sake of clarity, these are hereafter referred to as sheet (a) and sheet (b), respectively) (Fig. 6). Each ribbon is cnded at one side by a five-

Fig. 3. Stereoview of superposition of 10 refined solution structures of apo-neocarzinostatin. Backbone Ca, C, N, 0 atoms are shown. The structures are superimposed for minimum rms deviation between backbone atoms from residucs 4 to 113. The average structure is shown as the heavy line. The NHZ-and COOH-termini are indicated with yellow and red dot surfaces, respectively. Fig. 6. The average structure of apo-neocarzinostatinshown as a ribbon. The external layer of the seven-stranded /3-barrel is shown in blue, the internal Iayer in cyan. The two external loops, in the text referred as sheet (a) and sheet (b), are shown in white. Sheet (a) is closed by the disulfide bridge Cys37 - Cys47 indicated in yellow. The disulfide bridge Cys88 - Cys93 is shown in green. Sulfur atoms are indicated with space-filling representation. Fig. 7. cc-Carbon trace of the average structure of apo-neocarzinostatin which includes side chains of Phe52, Phe73, Phell2 and the two disulfide bridges. Fig. 8. Comparison of the solution structure of apo-neocarzinostatinwith that of the X-ray crystal structure of macromomycin 1171. The a-carbon trace and side-chain heavy atoms of apo-neocarzinostatin are shown in pink and in blue, respectively and the a-carbon trace and side-chain heavy atoms of macromomycin in white and in cyan, respectively. The residue numbering is that of apo-neocarzinostatin. Note that the aromatic residues of apo-neocarzinostatin (Tyr32, Phe73, Trp83 and Phel12) involved in the stabilization of the structure (see the text for details) are well superimposed with their corresponding residues in macromomycin.

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9 ("1 Fig. 4. Ramachandran 9, VJ plot for each amino acid residue of apo-neocarzinostatin. Only the average values for the 10 final refined structures are represented.

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30

40

50 60 70 Residue Number

80

90

100

110

Fig. 5. (A) Schematic diagram showing number of NOE conncctivities used in the final calculations versus residue number. (B, C) Average rms deviation from the mean for backbone (C) and side-chain (B) heavy atoms as a function of residue number for 10 structures, super-imposcd between residues 4 and 11 3 .

510 Table 3. Backbone-backbonehydrogen bonds formed in at least 5 of the 10 best structures of apo-neocarzinostatin. Donor

Acceptor

Frequency

20 Thr 0 111 Ser 0 69 Leu 0 69 Ser 0 67 Gly 0 63 Lys 0 61 G l y O 53 Ala 0 96 Tyr 0 94 Val 0 92 Gln 0 44 Cys 0 38 Gly 0 76 Val 0 32 Asp 0 30 Ser 0 23 Ala 0 21 Gly 0 19 Ala 0 17 Thr 0 86 Val 0 47 Ser 0 81 PheO 75 Asp 0 73 Arg 0 71 Gly 0 87 Asp 0 37 Thr 0 108 Ala 0 35 Ala 0 93 Pro 0

9 6 8 8 I0 8 8 5 8 7 10 6 9 10 9

~~

8 Thr N 13 Leu N 16 Gly N 17 T h r N 19 Val N 23 Gly N 26 Leu N 34 Vdl N 35 Gly N 37 Cys N 39 Trp N 40 Val N 46 Ala N 47 Cys N 55 Val N 57 Ala N 63 Ala N 65 Thr N 67 Leu N 69 Val N 73 Phe N 76 Phe N 77 Leu N 83 Trp N 86 Vdl N 88 Cys N 91 A l a N 94 Gln N 95 Val N 96 Gly N I20 Ilc N

10

9 8 10

9 8 5 6 9 5 10 I0 9 8 7 5

residue-containing bulged hairpin [33] involving residues 40 44 in sheet (a) and residues 77-81 in sheet (b). They are stabilized by an a-helical pattern of COi-NHi+4hydrogen bonds for Leu77 - Thr81 and for Val40 - Va144. The ribbons run almost orthogonal to each other and contacts between hydrophobic residues of the two sheets (Va138, Va140, Va144, Phe73, Leu77, Trp83 and Va186) maintain them close together. Main-chain hydrogen bonds between Cys47 and Phe76 also contribute in stabilizing the orthogonal packing. Sheet (a) is bounded to strand 5 of the barrel through Gln36, the side chain of which is buried into the interior of the barrel towards Arg70 and Arg71. This accounts for a significant broadening of the side-chain proton resonances for this residue. The other strand of this sheet is linked to strand 4 through a peptide segment running from residues Asp48 to Phe52. Residues 49-52 form a turn which detours around residues Gly35 and Gln36. Although this turn appears helical for residues 49 - 51 in the mean structure, it is poorly defined from the N M R data due to its probably large flexibility. Nevertheless, this turn inay contribute to the formation of a sulfur-aromatic interaction between Cys47 and Phe52 (Fig. 7) maintaining the protein stability [35]. A similar interaction involving Phe73 has been also reported [20] on the basis of Xray darn. In fdct, Phe73 points toward the disulfide bridge formed by Cys37 and Cys47 but lies too far away from the sulfur atoms (about 0.65 nm) to be implicated in a sulfur aromatic interaction (Fig. 7) [35]. A similar structural arrangement is found in macromomycin involving Cys36 - Cys46 and Phe72 (Fig. 8). The actual position of Phe.52, which has no

corresponding aromatic residue in macromomycin, seems to be more favorable for an association with this disulfide bridge. However, it cannot be excluded that Phe52 adopts a different conformation in the presence of the chromophore due to its position at the surface of the binding cleft. Finally, backbone hydrogen bonds between residues Cys37 and Gln94 on the one hand and Trp39 and Ala92 on the other hand help to pack sheet (a) towards the barrel. Sheet (b) is connected to strand 3 through residue Arg71 which has an helical conformation (average 4 = -60", average tp = -60") so that the polypeptide chain turns here by about 90". The other end of sheet (b) is bounded to strand 6 by a loop formed by the disulfide bridge between Cys88 and Cys93. This loop including residues 88 - 90 is helical as characterized by the 4 and yi angles and a COi-NHi+4hydrogen bond between Asp87 and Ala91. An hydrogen bond between Arg71 CO and Cys88 NH together with a sulfur aromatic interaction [35] between Phell2 and the two cysteines probably help to stabilize the orientation of the helix relative to the barrel. Phe132 is also in close contact with Arg71. In particular, the CclH of Arg7l lies above the ring of Phell2 accounting for its unusually high-field shifted resonance (2.26 ppm).

DISCUSSION AND CONCLUSIONS The present work confirms the great similarity of the overall structure of apo-neocarzinostatin, studied in solution, and of actinoxanthin [I61 and macromomycin [17] in the crystal. All these structures exhibit a well defined cleft, the putative binding site. For neocarzinostatin, this cavity is build up by four peptidic sequences comprising residues 32-48, 51 - 52, 73 - 82 and the large loop ranging over residues 97 - 107. The largest deviations with respect to the crystal structure of inacromomycin [17] concerns the loop between strands 6 and 7 involving residues 97- 107 (Fig. 8). Moreover, this loop exhibits the largest rms deviation indicating a large flexibility in solution. It could therefore adopt a different conformation upon chromophore binding. In fact, all the polypeptide segments involved in the cavity exhibit large rms deviations (Fig. 5). This is to be compared with the observation of higher thermal parameters for the corresponding regions in the crystal structure of macromomycin [17]. Despite a great structural similarity, the various proteins of the cytotoxic family exhibit a great selectivity with respect to their specific chromophore. Thus, this specificity should depend mainly, if not exclusively, upon the nature of the amino acid side chains forming the walls of the cleft characteristic of the family. Strikingly, the neocarzinostatin cavity includes five aromatic residues: Phe52, Phe73, Phe76, Phe78 and Trp39. This suggests that some of these residues play a role in stabilizing the complex by hydrophobic interactions between the aromatic part of the chromophore (Fig. 1) and the aromatic ring of the side chains. In fact, Trp39 has been previously shown by chemical modifications to be involved in the chromophore binding [36]. A direct investigation of the native protein could not directly answer this structural problem due to the great instability of the, chromophore under the conditions favorable for the NMR experiments. Fortunately, apo-neocarzinostatin binds daunomycin (Favaudon V., unpublished results), another antibiotic agent [37] which has structural elements very similar to those of the natural chromophore of neocarzinostatin, namely an anthraquinone aromatic ring and an

51 1 amino sugar. The proton-NMR spectra of this complex have been fully assigned by progressive titration from the apoprotein spectra. These results and their analysis in structural terms fully confirm the importance of hydrophobic binding of Phe52 and Phe78 in ligand stabilization, as will be reported in a forthcoming publication. We thank Dr. Valerie Biou and Prof. Jean Garnier of the Unite d’lngenierie des Protkines at the Institut National de la Recherche Agronomique for helpful discussions and encouragement. We gratefully acknowledge Prof. Patrick van Roey for providing us with the macromomycin coordinates before they were deposited within the Brookhaven Protein Data Bank.

Supplementary material

The coordinates of the final structures are available from the authors upon request.

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