Comparison of the independent solvent structures of dimeric alpha-chymotrypsin with themselves and with gamma-chymotrypsin

The solvent structure of alpha-chymotrypsin has been determined in the restrained least squares refinement (1.67-A resolution) of the dimeric molecule (Blevins, R. A., and Tulinsky, A. (1985) J. Biol. Chem. 260, 4264-4275). A total of 247 water molecules reduced the R-factor by 0.039 to 0.179. The average occupancy of solvent is 0.77 and the average isotropic thermal parameter is 22 A2. About 80% of the solvent is around the surface, 10% is in the dimer interface, and 10% is interior. There are 49 pairs of water molecules related by 2-fold noncrystallographic symmetry (within 1.0 A) and 199 waters that can potentially hydrogen bond with protein or themselves. The specificity sites contain 5 water molecules, 2 of which are displaced by substrate binding. The remainder probably aid in identifying and positioning the latter for catalysis. Four of these waters also occur in gamma-chymotrypsin. Considering the water structure in the dimer interface region of alpha-chymotrypsin with that of gamma-chymotrypsin reveals that about two-thirds of the solvent in this region is lost on dimerization. Last, 4 of the water molecules of alpha-chymotrypsin have been identified to be sulfate ions from a difference map based on crystals with selenate exchanged mother liquor.     

Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-A resolution

A 1.8-A resolution x-ray crystallographic restrained least squares refinement has been carried out on the phenylethane boronic acid (PEBA) complex of alpha-chymotrypsin dimer (alpha-CHT), and it has been compared to the 1.67-A resolution structure of the native enzyme. PEBA has a high binding affinity for alpha-CHT, and the boronate forms a tetrahedral complex with Ser-195 OG of one molecule of the dimer; the boronate in the other molecule is severely disordered and does not form a tetrahedral complex. The former could be a model of the transition state of catalysis. The complex of PEBA X alpha-CHT displays significant nonequivalence in conformation of side chains between the independent molecules comparable to the native enzyme, but, like the latter, shows a high degree of fidelity in the folding of the main chain. The orientation of the phenyl ring, CA and CB of PEBA, in the specificity sites of the two molecules is similar, suggesting that recognition is fairly insensitive to small departures from local symmetry; the same does not apply to the boronate functionalities suggesting that greater precision is required for catalysis. The folding of the molecule remains the same upon PEBA binding, but some of the side chains respond nonequivalently. The latter is a consequence of the inherent nonequivalence of the native dimer and the asymmetrical nature of the PEBA binding.     

Conformational folding of xyloglucan side chains in aqueous solution from molecular dynamics simulation

Molecular dynamics simulation was carried out on xyloglucan with explicit water molecules to investigate the folding mechanism of side chains onto a main chain in aqueous solution. The model xyloglucan was composed of 12 beta-D-glucopyranoses as a main chain substituted with six galactoses and three xyloses as side chains. Two conditions were set for the ribbon-like main chain; one is restricted to be 'flat' and the other is without restriction. The free main chain of xyloglucan has a 'twisted' conformation as the major one. Conformational folding of side chains onto the main chain was analyzed with dihedral angles at each glycosidic linkage. In a 5-ns calculation, the xyloglucan has a tendency to contract in both the restricted and the free systems, but the mode of contraction is different. Side chains tend to stick onto the flat surface of the main chain in the restricted system, while they do not tightly do so in the free one; instead the main chain takes a twisted and sometimes embowed conformation. This result indicates that the main chain has greater attractive forces to bind side chains when it is flat, while it loses the ability as it is twisted.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.     

Cooperativity during the formation of peptide/MHC class II complexes

To generate an effective immune response, class II major histocompatibility complex molecules (MHCII) must present a diverse array of peptide ligands for recognition by T lymphocytes. Peptide/MHCII complexes are stabilized by hydrophobic anchoring of peptide side chains to pockets in the MHCII protein and the formation of hydrogen bonds to the peptide backbone. Many current models of peptide/MHCII association assume an additive and independent contribution of the interactions between major MHCII pockets and corresponding side chains in the peptide. However, significant conformational rearrangements occur in both the peptide and MHCII during binding. Therefore, we hypothesize that peptide binding to MHCII could be viewed as a folding process in which both molecules cooperate to produce the final conformation. To directly test this hypothesis, we adapt a serial mutagenesis strategy to study cooperativity in the interaction of the human MHCII HLA-DR1 and a peptide derived from influenza hemagglutinin. Substitutions in either the peptide or HLA-DR1 that are predicted to interfere with hydrogen bond formation show cooperative effects on complex stability and affinity. Substitution of a peptide side chain that provides a hydrophobic contact also contributes to the cooperative effect, suggesting a role for all energetic sources in the folding process. We propose that cooperativity throughout the peptide-binding groove reflects the folding of segments of the MHCII molecule into helices around the peptide with a concomitant folding of the peptide into a polyproline helix. The implications of cooperativity for peptide/MHCII structure and epitope selection are discussed.     

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.     

Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution

The molecular structure of the complex between bovine pancreatic alpha-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I greater than or equal to sigma (I)]. The reactive site loop of OMTKY3, from Lys13I to Arg21I (I indicates inhibitor), is highly complementary to the surface of alpha-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from O gamma of Ser195 to the carbonyl-carbon atom of the scissile bond between Leu18I and Glu19I is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P'3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the alpha-chymotrypsin portion of this complex with the several structures of alpha and gamma-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common alpha-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central alpha-helix and flanking small 3-stranded beta-sheet) that have alpha-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (C alpha of Leu18I). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and alpha-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.     

Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.     

Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution

The crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 A resolution. While the wild-type CGTase from the same bacterium produces a mixture of mainly alpha-, beta- and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains, site-directed mutation of histidine-233 to asparagine changed the nature of the enzyme such that it no longer produced alpha-cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two-fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild-type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine-233. The differences in hydrogen bonds in the neighborhood of asparagine-233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase.     

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.     

Differential scanning calorimetry study of the hydrophobic hydration of the elastin-based polypentapeptide, poly(VPGVG), from deficiency to excess of water

The polypentapeptide of elastin, poly(VPGVG), has become an interesting model polypeptide in understanding the mechanism of protein folding and assembly. Due to its simple amino acid composition and the predominance of apolar side chains, this polymer shows strong hydrophobic-hydration phenomena. This paper explores, by calorimetric methods, the nature and structure of the clathrate-like arrangements that take place, surrounding the apolar side chains of the polymer. The performance of these methods, especially differential scanning calorimetry, has a well-gained reputation. In this work, the development of the clathrate-like structures around this model polymer has been followed from water deficiency to water-excess states. Two main conclusions have been obtained from the data obtained. First, there is an upper limit of about 170 water molecules per pentamer as the number of water molecules required to form all the possible clathrate-like structures. Second, these structures exist as an inhomogeneous population with energies spreading in a significantly broad range, which is likely related to differences in geometrical parameters (bond lengths and angles) of the clathrate structure.Copyright 2000 John Wiley & Sons, Inc.     

Measurement and computation of the dipole moment of globular proteins III: Chymotrypsin

The dipole moments of alpha- and gamma-chymotrypsin are determined experimentally using the dielectric constant measuring method. The values thus obtained are compared with the results of the electric dichroism measurements for alpha-chymotrypsins by other investigators. The agreement is reasonably good, if not satisfactory. The cause of difference appears to be due to the difficulty of finding the correct internal field. The interaction between two neighboring dipoles is found to be a minor component of the local fields. Secondly, the dipole moment of alpha-chymotrypsin was computed using Protein Data Bases. The dipole moment of proteins consists of two major components, the moment due to fixed surface charges and the core moment due to polar chemical bonds. The method of calculation was described in detail in previous papers. The pK shifts of polar side chains were calculated using the methods of Tanford et al. and its modification by Warshel et al. The agreement between measured and calculated dipole moments is satisfactory.     

Structural basis of the catalytic role of Glu301 in Anabaena PCC 7119 ferredoxin-NADP+ reductase revealed by x-ray crystallography

The three-dimensional crystal structure of the Glu301Ala site-directed mutant of ferredoxin-NADP+ reductase from Anabaena PCC 7119 has been determined at 1.8A resolution by x-ray diffraction. The overall folding of the Glu301Ala FNR mutant shows no significant differences with respect to that of the wild-type enzyme. However, interesting conformational changes are detected in the side chain of another glutamate residue, Glu139, which now points towards the FAD cofactor in the active center cavity. The new conformation of the Glu139 side chain is stabilized by a network of five hydrogen bonds to several water molecules, which seem to hold the carboxylate side chain in a rather fixed position. This interacting network connects the Glu139 side chain to the Ser80 side chain through a series of three water molecules. These observations are discussed in terms of the reactivity of Glu301Ala ferredoxin-NADP+ reductase towards its substrates, and the role of Glu301 in the catalysis is re-examined. Moreover, a structural explanation of the different reoxidation properties of this mutant is given on the basis of the reported structure by modeling the hypothetical flavin C(4a)-hydroperoxide intermediate. The model shows that the distal oxygen of the peroxide anion could be in an appropriate situation to act as the proton donor in the reoxidation process.     

Lateral chain packing in lipids and membranes

The aliphatic chains of many biologically important lipids are heterogeneous and often related to the functions of the molecules. Certain phospholipids containing arachidonic acid may serve as precursors for prostaglandins, certain diglycerides may serve as second messengers for certain membrane-triggered reactions (43), and other phospholipids containing a very short chain in the two position may serve as vasoactive hormones (44). The packing of such molecules is of interest. The evidence is quite clear from both the conformation of saturated and unsaturated molecules and from mixing experiments in the solid state that long and short chains don't mix well, nor do unsaturated and saturated chains, even if they are of the same chain length. There is even some evidence to indicate that some degree of chain segregation occurs even in the liquid state. However, different chains are often associated through covalent bonds, e.g., in wax esters, diacylglycerols, triacylglycerols, and phospholipids. A variety of possibilities for chain segregation are present in the neat phases of wax esters, ceramides, diacylglycerols, and triacylglycerols. However, in the unique case of membrane lipids like phospholipids or sphingolipids, the two chains are forced to lie side by side by virtue of the interaction of the polar group with water, and thus interactions between different chains must occur. Most of the evidence suggests that, when a solid phase results in these systems, the nonspecific chain packing mode (hexagonal chain packing) is preferred. In fact, for all of the phospholipids studied thus far, clearcut evidence of specific chain-chain interaction in molecules having both unsaturated and saturated chains has never been observed. However, for mixed chain triacylglycerols, evidence of specific chain-chain interactions (beta' and even beta) has been found and some suggestions have been given as to how this might occur through chain segregation mechanisms in the neat state. The literature suggests that further work needs to be done on the interaction of different chains that are covalently linked to the same molecule. Such studies will lead to a better understanding of the structure of lipid bilayers, membranes, lipoproteins, and lipid deposits.     

Hierarchical protein folding: asymmetric unfolding of an insulin analogue lacking the A7-B7 interchain disulfide bridge

The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (Cys(A7)-Cys(B7)) are characterized as a model of a late intermediate. Untethering cystine A7-B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6-A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20-B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains native-like supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.     

Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein.

The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an alphahelix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain chi1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.     

Mimetics of a T cell epitope based on poly-N-acylated amine backbone structures induce T cells in vitro and in vivo

Peptidomimetics of the major histocompatibility complex (MHC) class I-restricted ovalbumin-derived T cell epitope SIINFEKL were generated by replacing parts of the peptide backbone by a poly-N-acylated amine (PAA) backbone with aromatic, heteroaromatic, and pseudoaromatic side chains that branch off of the main chain at the amine nitrogen. The structure of the PAAs was designed to position this side chain in the central epitope anchor pocket of the MHC molecule. A number of biologically active PAAs were found that induced cytolysis by the mouse cytotoxic T cell clone 4G3. Competition experiments with independent peptides that are known to bind to the restricting MHC molecule H-2K(b) suggest that the PAAs are bound by the MHC molecules at the same site as conventional peptide epitopes. The PAAs were active also in vivo and induced primary cytotoxic T cell responses in mice.     

Organic solvent changes the chymotrypsin specificity with respect to nucleophiles.

In alpha-chymotrypsin-catalyzed acyl-transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of alpha-chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium.     

Dynamic polyhedral models of globular proteins

We have devised several mechanical models of globular proteins by approximating them to various polyhedra (dodecahedron, truncated octahedron, icosahedron, truncated icosahedron). The models comprise hollow blocks linked together in a flexible chain. Between blocks there is a set of several reversible, weak magnetic interactions such that when the chain is agitated, it will fold into a stable polyhedral structure about the size of a hand. Folding may be followed in real time with a video camera. Key to the success of the folding process is the lightness of the chain. Several side chains may also be added to the blocks such that they come together to create a polyhedral core when the chain folds. The models have a number of similarities to globular proteins: each chain folds into a unique, but dynamic, three-dimensional structure; the instructions that determine this structure are built into the configuration of blocks; and it is difficult to predict this structure given the unfolded block configuration. Furthermore, the chains fold quickly, generally in less than a minute, several pathways are involved, and these pathways progress through elements of "native" structure. In particular, the models emphasize the importance of restricted conformational mobility in assisting the chain to fold, and also in eliminating undesirable interactions. Because of these similarities to globular proteins, we believe that the polyhedral models will, with continued development, be helpful in understanding the protein folding process, while at the same time acting as valuable educational visual aids. They might also inspire the construction of new types of microscopic, self-assembling devices.     

The three-dimensional structure of the 4:1 mithramycin:d(ACCCGGGT)(2) complex: evidence for an interaction between the E saccharides

Mithramycin and chromomycin, two antitumor drugs, each having an identical aglycone and nearly identical disaccharide and trisaccharide side chains, have differing binding properties to a small oligonucleotide, d(ACCCGGGT)(2) (M. A. Keniry et al., Journal of Molecular Biology, 1993, Vol. 231, pp. 753-767). In order to understand the forces that induce four mithramycin molecules to bind to d(ACCCGGGT)(2) instead of two drug molecules in the case of chromomycin, the structure of the 4:2:1 mithramycin: Mg(2+):d(ACCCGGGT)(2) complex was investigated by (1)H-nmr and restrained molecular dynamics. The resulting three-dimensional model showed that in order to accommodate the close approach of one neighboring mithramycin dimer, the inwardly directed CDE saccharide chain of the neighboring mithramycin dimer undergoes a conformational change such that the E saccharide no longer spans the minor groove but reorients so that the hydrophilic face of the E saccharides from the two dimers oppose each other. Two hydrogen bonds are formed between the hydroxyl groups of the two opposing E saccharide groups. The results are interpreted in terms of the differences in stereochemistry and functional group substitutions between mithramycin and chromomycin. A mithramycin dimer is able to self-associate on an oligonucleotide template because it has two hydroxyl groups on the same face of its terminal E saccharide. A chromomycin dimer is unable to self-associate because one of these hydroxyl groups is acetylated and the neighboring hydroxyl group has a stereochemistry that cannot permit close contact of the hydroxyl group with a neighbouring chromomycin dimer.Copyright 2000 John Wiley & Sons, Inc.