Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography.

Non-specific lipid-transfer proteins (nsLTPs) are involved in the movement of phospholipids, glycolipids, fatty acids, and steroids between membranes. Several structures of plant nsLTPs have been determined both by X-ray crystallography and nuclear magnetic resonance. However, the detailed structural basis of the non-specific binding of hydrophobic ligands by nsLTPs is still poorly understood. In order to gain a better understanding of the structural basis of the non-specific binding of hydrophobic ligands by nsLTPs and to investigate the plasticity of the fatty acid binding cavity in nsLTPs, seven high-resolution (between 1.3 A and 1.9 A) crystal structures have been determined. These depict the nsLTP from maize seedlings in complex with an array of fatty acids.A detailed comparison of the structures of maize nsLTP in complex with various ligands reveals a new binding mode in an nsLTP-oleate complex which has not been seen before. Furthermore, in the caprate complex, the ligand binds to the protein cavity in two orientations with equal occupancy. The volume of the hydrophobic cavity in the nsLTP from maize shows some variation depending on the size of the bound ligands. The structural plasticity of the ligand binding cavity and the predominant involvement of non-specific van der Waals interactions with the hydrophobic tail of the ligands provide a structural explanation for the non-specificity of maize nsLTP. The hydrophobic cavity accommodates various ligands from C10 to C18. The C18:1 ricinoleate with its hydroxyl group hydrogen bonding to Ala68 possibly mimics cutin monomer binding which is of biological importance. Some of the myristate binding sites in human serum albumin resemble the maize nsLTP, implying the importance of a helical bundle in accommodating the non-specific binding of fatty acids.

RNase activity of a DNA minor groove binder with a minimalist catalytic motif from RNase A.

Imidazole and compounds containing imidazole residues have been shown to cleave RNA in an RNase A-mimicking manner. Di-imidazole lexitropsin is a compound which is derived from the polyamide drugs distamycin and netropsin essentially by the replacement of two pyrrole heterocycles with N-methyl-imidazole residues. This enables it to bind to the minor groove of B-DNA in a sequence-specific manner. We demonstrate here that this lexitropsin derivative has RNA cleavage activity, as tested on model RNAs. Optimal cleavage conditions and cleavage specificity resemble those known from other imidazole conjugates and are thus consistent with an RNase A type cleavage mechanism. The optimum concentration of the compound for cleavage is similar to previously investigated imidazole-based RNase mimics. As a whole new class of chemical compounds capable of interacting with nucleic acids through extensive hydrogen bonding, these imidazole containing compounds constitute promising scaffolds and ligands, for the construction of novel RNase mimics with high affinity.

The structure of a stable intermediate in the A <--> B DNA helix transition.

The DNA dodecamer CATGGGCCCATG in a crystal structure of resolution 1.3 A has a conformation intermediate between A and B DNA. This trapping of a stable intermediate suggests that the A and B DNA families are not discrete, as previously believed. The structure supports a base-centered rather than a backbone-centered mechanism for the A <--> B transition mediated by guanine tracts. Interconversion between A and B DNA provides another means for regulating protein-DNA recognition.

Defining GC-specificity in the minor groove: side-by-side binding of the di-imidazole lexitropsin to C-A-T-G-G-C-C-A-T-G.

BACKGROUND: Polyamide drugs, such as netropsin, distamycin and their lexitropsin derivatives, can be inserted into a narrow B-DNA minor groove to form 1:1 complexes that can distinguish AT base pairs from GC, but cannot detect end-for-end base-pair reversals such as TA for AT. In contrast, 2:1 side-by-side polyamide drug complexes potentially are capable of such discrimination. Imidazole (Im) and pyrrole (Py) rings side-by-side read a GC base pair with the Im ring recognizing the guanine side. But the reason for this specific G-Im association is unclear because the guanine NH2 group sits in the center of the groove. A 2:1 drug:DNA complex that presents Im at both ends of a GC base pair should help unscramble the issue of imidazole reading specificity. RESULTS: We have determined the crystal structure of a 2:1 complex of a di-imidazole lexitropsin (DIM), an analogue of distamycin, and a DNA decamer with the sequence C-A-T-G-G-C-C-A-T-G. The two DIM molecules sit antiparallel to one another in a broad minor groove, with their cationic tails widely separated. Im rings of one drug molecule stack against amide groups of the other. DIM1 rests against nucleotides C7A8T9G10 of strand 1 of the helix, whereas DIM2 rests against G14G15C16C17 on strand 2. All DIM amide nitrogens donate hydrogen bonds to N and O atoms on the floor of the DNA groove and, in addition, the two Im rings on DIM2 accept hydrogen bonds from guanine N2 amines, thereby providing specific reading. The guanine N2 amine can bond to Im on its own side of the groove, but not on the cytosine side, because of limits on close approach of the two Im rings and the geometry of sp2 hybridization about the amide nitrogen. CONCLUSIONS: Im and Py rings distinguish AT from GC base pairs because of steric factors involving the bulk of the guanine amine, and the ability of Im to form a hydrogen bond with the amine. Side-by-side Im and Py rings differentiate GC from CG base pairs because of tight steric contacts and sp2 hybridization at the amine nitrogen atom, with the favored conformations being G/Im,Py/C and C/Py,Im/G. Discrimination between AT and TA base pairs may be possible using bulkier rings, such as thiazole to select the A end of the base pair.

Structure of a DNA analog of the primer for HIV-1 RT second strand synthesis.

The non-self-complementary DNA decamer C-A-A-A-G-A-A-A-A-G/C-T-T-T-T-C-T-T-T-G is a DNA/DNA analogue of a portion of the polypurine tract or PPT, which is a RNA/DNA hybrid that serves as a primer for synthesis of the (+) DNA strand by HIV reverse transcriptase (RT), and which is not digested by the RNase H domain of reverse transcriptase following (-) strand synthesis. The same unusual conformation that eludes RNase H, thought to be a change in width of minor groove, may also be responsible for the inhibition of HIV RT by minor groove binding drugs such as distamycin and their bis-linked derivatives. The present X-ray crystal structure of this DNA decamer exhibits the usual properties of A-tract B-DNA under biologically relevant conditions: large propeller twist of base-pairs, narrowed minor groove, and a straight helix axis. Groove narrowing is fully developed in the A-A-A-A region, but not in the A-A-A region, which previous investigators have proposed as being too short to exhibit typical A-tract properties. The RNA/DNA hybrid produced by HIV reverse transcriptase during (-) strand synthesis presumably forms a "heteromerous" or H-helix with narrower minor groove than an A-helical RNA/RNA duplex. If the narrowing of minor groove in A-tract H-helices is comparable to that seen in A-tract B-helices, then the narrowed minor groove of the polypurine tract could make the second primer site both (1) impervious to RNase H digestion, and (2) susceptible to inhibition by minor groove binding drugs.

Progress in the design of DNA sequence-specific lexitropsins.

Sequence-specific polyamides that bind in the minor groove of DNA are attractive candidates for antibiotics, cancer chemotherapeutics, and transcriptional antagonists. This paper reviews the progress of structure-based design of minor-groove-binding polyamides, from the first structure of netropsin with DNA, to the effective linked polyamides currently under study. A theory of polyamide specificity is also reviewed, introducing methods to determine the optimal strategies for targeting a given DNA sequence within a genome of competing sequences.

Design of stapled DNA-minor-groove-binding molecules with a mutable atom simulated annealing method.

We report the design of optimal linker geometries for the synthesis of stapled DNA-minor-groove-binding molecules. Netropsin, distamycin, and lexitropsins bind side-by-side to mixed-sequence DNA and offer an opportunity for the design of sequence-reading molecules. Stapled molecules, with two molecules covalently linked side-by-side, provide entropic gains and restrain the position of one molecule relative to its neighbor. Using a free-atom simulated annealing technique combined with a discrete mutable atom definition, optimal lengths and atomic composition for covalent linkages are determined, and a novel hydrogen bond 'zipper' is proposed to phase two molecules accurately side-by-side.

Linked lexitropsins and the in vitro inhibition of HIV-1 reverse transcriptase RNA-directed DNA polymerization: a novel induced-fit of 3,5 m-pyridyl bisdistamycin to enzyme-associated template-primer.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

MPD and DNA bending in crystals and in solution.

Bending of 15 to 24 degrees is observed within crystal structures of B-DNA duplexes, is strongly sequence-dependent, and exhibits no correlation with the concentration of MPD (2-methyl-2,4-pentanediol) in the crystallizing solution. Two types of bends are observed: facultative bends or flexible hinges at junctions between regions of G.C and A.T base-pairs, and a persistent and almost obligatory bend at the center of the sequence R-G-C-Y. Only A-tracts are characteristically straight and unbent in every crystal structure examined to date. A detailed examination of normal vector plots for individual strands of a double helix provides an explanation, in terms of the stacking properties of guanine and adenine bases. The effect of high MPD concentrations, in both solution and crystal, is to decrease local bending somewhat without removing it altogether. MPD gel retardation experiments provide no basis for choosing among the three models that seek to explain macroscopic curvature of DNA by means of microscopic bending: junction being, bent A-tracts, or bent general -sequence DNA. Crystallographic data on the straightness of A-tracts, the bendability of non-A sequences, and the identity of inclination angles in A-tract and non-A-tracts B-DNA support only the general-sequence bending model. The pre-melting transition observed in A-tract DNA probably represents a relaxation of stiff adenine stacks to a flexible conformation more typical of general-sequence DNA.

Structure of a dicationic monoimidazole lexitropsin bound to DNA.

An X-ray crystal structure has been solved of the complex of a dicationic lexitropsin with a B-DNA duplex of sequence CGCGAATTCGCG. The lexitropsin is identical to netropsin except for replacement of the first methylpyrrole ring by methylimidazole, converting a =CH- to =N-. Crystals are isomorphous with those of the DNA dodecamer in the absence of drug. Although the =N- for =CH- substitution was intended to make that locus on the drug molecule compatible with a G.C base pair, electrostatic attraction for the two cationic ends of the drug predominates, and this lexitropsin binds to the same central AATT site as does the parent netropsin. But unlike netropsin, this lexitropsin exhibits end-for-end disorder in the crystal. Both orientations were refined separately to completion. Final residual errors at 2.25 A resolution for the 2358 reflections above 2 sigma in F are R = 0.165 for one orientation (LexA) with 37 water molecules and 0.164 for the inverted drug orientation (LexB) with 40 water molecules. This molecular disorder is probably attributable to a weakening of binding to the AATT site occasioned by the imidazole-for-pyrrole substitution.

Design of B-DNA cross-linking and sequence-reading molecules.

We report the design of hybrid molecules to bind in the minor groove of B-DNA, which combine DNA alkylating and cross-linking ability for increased chemotherapeutic efficacy, with sequence specificity, to minimize side effects. Optimal linkage geometries have been determined for the synthesis of bis-anthramycin and anthramycin-netropsin hybrid molecules. Earlier studies on linked drugs have typically been based on molecular mechanics calculations. This work, in contrast, uses the observed crystal structures of a netropsin/DNA complex and a new anthramycin/DNA complex to determine the exact spacing between two individual drugs when bound in the minor groove of B-DNA. Molecular linkers then are designed and tested between these two experimental positions, to form a chimeric or bis-linked compound molecule. A linked anthramycin-netropsin molecule has been designed specifically to target the polypurine tract second-strand primer site of the reverse transcriptase of HIV-1.

Refinement of netropsin bound to DNA: bias and feedback in electron density map interpretation.

The X-ray crystal structure of the complex of the B-DNA dodecamer CGCGAATTCGCG with the antitumor drug netropsin has been reexamined to locate the drug accurately for computer-based drug design. The optimum solution is with the drug centered in the AATT region of the minor groove, making three good bifurcated hydrogen bonds with adenine N3 and thymine O2 atoms along the floor of the groove. Pyrrole rings of netropsin are packed against the C2 positions of adenines, leaving no room for the amine group of guanine and, hence, providing a structural rationale for the A.T specificity of netropsin. An alternative positioning in which the drug is shifted along the minor groove by ca. one-half base pair step is rejected on the basis of free R factor calculations and the appearance of the original drug-free difference maps. Final omit maps, although of more pleasing appearance, are not a dependable means of discriminating between right and wrong structures. The shifted alternative drug position ignores potential hydrogen bonding along the floor of the groove, provides no explanation for netropsin's observed A.T specificity, and is contradicted by NMR results [Patel, D. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6424].

Crystal structure of a covalent DNA-drug adduct: anthramycin bound to C-C-A-A-C-G-T-T-G-G and a molecular explanation of specificity.

A 2.3-A X-ray crystal structure analysis has been carried out on the antitumor drug anthramycin, covalently bound to a ten base pair DNA double helix of sequence C-C-A-A-C-G-T-T-G-G. One drug molecule sits within the minor groove at each end of the helix, covalently bound through its C11 position to the N2 amine of the penultimate guanine of the chain. The stereochemical conformation is C11S, C11aS. The natural twist of the anthramycin molecule in the C11aS conformation matches the twist of the minor groove as it winds along the helix; a C11aR drug would only fit into a left-handed helix. The C11S attachment is roughly equatorial to the overall plane of the molecule, whereas a C11R attachment would be axial and would obstruct the fitting of the drug into the groove. The six-membered ring of anthramycin points toward the 3' end of the chain to which it is covalently attached or toward the end of the helix. The acrylamide tail attached to the five-membered ring extends back along the minor groove toward the center of the helix, binding in a manner reminiscent of netropsin or distamycin. The drug-DNA complex is stabilized by hydrogen bonds from C9-OH, N10, and the end of the acrylamide tail to base pair edges on the floor of the minor groove. The origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds but from the low twist angles adopted by purine-purine steps in a B-DNA helix. Binding of anthramycin induces a low twist at T-G in the T-G-G sequence of this DNA-drug complex, by comparison with the structure of the free DNA. The origin of anthramycin's preference for adenines flanking the alkylated guanine arises from a netropsin-like fitting of the acrylamide tail into the minor groove.

Crystal structure of CATGGCCATG and its implications for A-tract bending models.

The single-crystal x-ray analysis of orthorhombic CATGGCCATG has revealed a previously unrecognized mode of intrinsic bending in DNA. The decamer shows a smooth bend of 23 degrees over the central four base pairs, caused by preferential stacking interactions at guanine bases. The bend is produced by a roll of base pairs along their long axes, in a direction that compresses the wide major groove of the double helix. This major-groove-compressing bend at GGC, plus the abundant crystallographic evidence that runs of successive adenine bases (A-tracts) are straight and unbent, requires rethinking of the models most commonly invoked to explain A-tract bending. A decade of excellent experimental work involving gel migration experiments, cyclization kinetics, and nucleosome phasing has clearly established that introduction of short A-tracts into a general DNA sequence in synchrony with the natural repeat of the helix leads to bending. But it does not logically and inevitably follow that the actual bending is to be found within these introduced A-tracts or even at junctions with general-sequence B-DNA.

Structure of the B-DNA decamer C-C-A-A-C-I-T-T-G-G in two different space groups: conformational flexibility of B-DNA.

For the first time, the same B-DNA oligomer has been crystallized and its structure solved in two different space groups. Crystallization of C-C-A-A-C-I-T-T-G-G with Ca2+ yields monoclinic space group C2 with a = 31.87 A, b = 25.69 A, c = 34.21 A, beta = 114.1 degrees, and five base pairs per asymmetric unit. The 5026 2 sigma data to 1.3 A refine to R = 0.152 with 72 waters, one heptavalent hydrated calcium complex, and one cacodylate ion per asymmetric unit. In contrast, crystallization with Mg2+ yields trigonal space group P3(2)21 with a = b = 33.23 A, c = 94.77 A, gamma = 120 degrees, and 10 base pairs per asymmetric unit. The 1725 2 sigma data to 2.2 A refine to R = 0.164 with 36 water molecules and one octahedral magnesium complex per asymmetric unit. The monoclinic form is virtually isostructural with previously solved monoclinic decamers, including twist angles of ca. 50 degrees at C-A and T-G steps. In contrast, the trigonal structure has quite different local helix parameters, with twist angles of ca. 36 degrees at the corresponding steps. These local parameter differences can only be attributed to crystal packing, suggesting that certain sequences of B-DNA are more flexible and influenced by their surroundings than had previously been thought. Such deformability may be important for interaction of B-DNA with control proteins, where both static structure and dynamic deformability comprise components of the recognition process. The crossing of two helices at an angle of 120 degrees in the trigonal cell is a model for an antiparallel, uncrossed Holliday junction, as has been noted earlier by Timsit and Moras [Timsit, Y., & Moras, D. (1991) J. Mol. Biol. 221, 919-940] from a rhombohedral DNA dodecamer structure analysis.

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.

The effect of crystal packing on oligonucleotide double helix structure.

One of the questions that constantly is asked regarding x-ray crystal structure analyses of macromolecules is: To what extent is the observed crystal structure representative of the molecular conformation when free in solution, and to what degree is the structure perturbed by intermolecular crystal forces? This can be assessed with DNA oligomers because of an unusual aspect of crystallization self-complementary oligomers should possess a twofold symmetry axis normal to their helix axis, yet more often than not crystal of such oligomers do not use this internal symmetry. The two ends of the helix are crystallographically distinct though chemically identical. Complexes of DNA oligomers with intercalating drugs such as triostin A tend to use their twofold symmetry when they crystallize, whereas complexes with non-intercalating, groove-binding drugs ignore this symmetry unless the drug molecule is very small. A detailed examination of crystal packing in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G provides an explanation of all of the foregoing behavior in terms of the mechanism of nucleation of DNA or DNA-drug complexes on the surface of a growing crystal. Asymmetry of the ends of the DNA helix is the price that is paid for efficient lateral packing of helices within the crystal. The actual end-for-end variation in standard helix parameters is compared with the experimental noise level as gauged by independent re-refinement of the same oligonucleotide structure where available, and with the observed extent of variation of these same parameters along the helix. Oligomers analyzed are the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, the A-DNA octamer G-G-T-A-T-A-C-C, and the phosphorothioate analogue of the B-DNA hexamer G-C-G-C-G-C. End-for-end variation, presumably the result of crystal packing is typically double the experimental noise level, and half the variation in the same parameter along the helix. Analysis of crystal packing in the phosphorothioate hexamer, which uses the same P212121 space group as the dodecamer, shows that the highly unsymmetrical B1 vs. BII backbone conformation probably is to be ascribed to crystal packing forces, and not to the sequence of the hexamer.

Helix geometry, hydration, and G.A mismatch in a B-DNA decamer.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

Nuclear Overhauser data and stereochemical considerations suggest that netropsin binds symmetrically within the minor groove of poly(dA).poly(dT), forming hydrogen bonds with both strands of the double helix.

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA).poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982] and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985] are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G.

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.