Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1870-1873, March 1990 Biochemistry lac repressor: Crystallization of intact tetramer and its complexes with inducer and operator DNA (Escherchia coli lactse operon/gene reguation/aflostery/crystallography) HELEN C. PACEt, PONZY Lut, AND MITCHELL LEWISt*§ tDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104; and tMacromolecular Sciences Department, Smith Kline & French Laboratories, King of Prussia, PA 19406 Communicated by Peter H. von Hippel, December 11, 1989 ABSTRACT The intact kac repressor tetramer, which reg- near the DNA while the second helix fits into the major ulates expression ofthe kac operon in Escherichia coli, has been groove and provides the majority of the sequence-specific crystallized in the native form, with an inducer, and in a interactions. Sequence comparisons have indicated that ternary complex with operator DNA and an anti-inducer. The many other DNA-binding proteins may also contain such crystals without DNA diffract to better than 3.5 A. They belong bihelical units (25-28). Solution NMR studies (29, 30) suggest to the monocinic space group C2 and have cell dimensions a = that the lac repressor also has a helix-turn-helix at the N 164.7 A, b = 75.6 A, and c = 161.2 i, with a = Y = 90° and terminus. (3 = 125.5°. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal MATERIALS AND METHODS bipyramids that diffract to 6.5 A. These protein-DNA cocrys- Isolation of lac Repressor. The production of lac repressor tals crack upon exposure to the gratuitous inducer isopropyl was accomplished by using an expression system with a (J-D-thiogalactoside, suggesting a conformational change in the multicopy plasmid, pIQ (31), in a GM1 strain of E. coli (32). repressor-operator complex. The isolation protocol was based on published procedures (33-35), with the following changes: DNase was not used in The lactose operon of Escherichia coli is the paradigm for the lysing buffer, but up to 0.2 mg of phenylmethylsulfonyl gene regulation (1-7). lac repressor, the tetrameric protein fluoride, a protease inhibitor, and 0.2 mg of lysozyme per ml that controls expression of the operon genes, has been ofbuffer to compensate for the absence ofA lysozyme, which studied by genetic, biochemical, and physical methods for was present in the previous procedure, were used; sonication nearly 30 years (1, 2). However, a complete understanding of was used to break cells and fragment genomic DNA. In this protein requires knowledge of its three-dimensional addition, our protocol included fast protein liquid chroma- structure. Only then may we address the question ofhow this tography (Pharmacia). This involved gel filtration using Su- repressor recognizes and binds to a specific DNA site and perose 6 medium (Pharmacia) followed by cation exchange responds to effector molecules to regulate gene expression. over a Mono S column (Pharmacia). For some of the exper- We have begun a crystallographic investigation of the intact iments, Amicon Centricon 30 units were used to concentrate lac repressor and its complexes with an inducer and with the repressor to about 40 mg/ml. operator DNA. Purification of Synthetic Oligonucleotides. Operator DNA lac repressor is the product of the I gene of E. coli. It fragments were synthesized on an Applied Biosystems model consists offour identical subunits containing 360 amino acids 380A DNA synthesizer or a Milligen/Biosearch Cyclone by of known sequence (8, 9), with a total molecular weight of the solid-phase phosphoramidite method (36). Dimethoxytri- 154,520. This protein is particularly interesting because it is tyl-bearing fragments were purified by HPLC over a Beck- tetrameric, unlike the other DNA-binding proteins for which man C18 column to separate out failure fragments and ben- structures have been reported (10-21). It appears to interact zamide (a by-product of the synthesis). After the dimethoxy- with looped DNA at two sites (22-24) and is postulated to be trityl group was removed, a second HPLC purification was an allosteric protein (7). used to resolve the desired peak from n - 1 fragments. A The three-dimensional structures of several intact regula- volatile buffer (0.1 M ammonium bicarbonate, pH 7.8, with tory proteins and the DNA-binding fragments of two others an acetonitrile gradient) was used, which allowed evapora- have been determined by x-ray analysis. They include the tion of the product to dryness without desalting. Single DNA-binding domain of the cI repressors and the complete strands were annealed, and the duplexes were stored at cro repressors from bacteriophages A (10, 11) and i434 (12, -700C in dry aliquots. They were directly dissolved into 13), the catabolite gene activator protein (CAP) (14), the crystallization buffer as needed. tryptophan repressor (15), and the met repressor (55) from E. coli. With the exception of CAP, the structures of these proteins complexed with DNA have also been reported RESULTS (16-21). These proteins regulate the expression of specific Crystallization, Space Group, and Unit Cell Dimensions. lac genes by either preventing or enhancing the level of tran- repressor native tetramer. The crystallization buffer was scription at DNA sites near the promoter. Despite their similar to that used by Steitz et al. (37) to obtain microcrystals differences in size and tertiary structure, they bind to DNA with isopropyl (3-D-thiogalactoside (IPTG). Crystals were in a common fashion. Two a-helices, linked by a tight turn, grown without IPTG for 3-5 months at room temperature by are conserved among all these regulatory proteins. In this hanging-drop vapor diffusion. Seeding was employed to helix-turn-helix motif, the first helix sits above the groove Abbreviation: IPTG, isopropyl /-D-thiogalactoside. The publication costs ofthis article were defrayed in part by page charge §To whom reprint requests should be addressed at his present payment. This article must therefore be hereby marked "advertisement" address: Department of Biochemistry and Biophysics, School of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicine, University of Pennsylvania, Philadelphia, PA 19104. 1870 Downloaded by guest on September 29, 2021 Biochemistry: Pace et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1871 FIG. 1. lac repressor crystals alone (Left), with IPTG (Center), and with 16-bp operator DNA (Right). The longest dimensions of crystals shown are 0.5 mm (Left), 0.3 mm (Center), and 1.3 mm (Right). increase size and decrease growth time. Numerous different native repressor. IPTG concentrations ranging from 1 to 4 morphologies were found, including flat, six-sided plates times the number of repressor binding sites were used. measuring about 0.5 x 0.25 x 0.1 mm. Crystals of various Though these crystals appear to have nicer shapes with shapes are shown in Fig. 1 Left. smoother faces than those grown in the absence of IPTG, a The native tetramer crystals belong to the space group C2 variety of morphologies are still seen (Fig. 1 Center). Pre- as determined by precession photography (Fig. 2). This cession photographs show that they are isomorphous with the monoclinic crystal form has cell dimensions a = 164.7 A, b native crystals, with no apparent intensity differences at low = 75.6 A, and c = 161.2 A, with a = y = 90° and 8 = 125.5°, resolution. with a unit cell volume of 1.63 x 106 A3. Based upon an lac repressor-lac operator DNA cocrystals. The wild-type average Matthews coefficient (38) of 2.6 for a protein of this lac operator sequence of about 30 base pairs (bp), like many size, we would expect that the crystals contain 54% solvent. operators, is almost twofold symmetric (39). We have This would result in one tetramer per asymmetric unit (four screened five different lengths ofthe symmetric lac operator- tetramers per unit cell). related sequences shown in Fig. 3. These DNA fragments Oscillation photos taken at the Stanford Synchrotron Ra- were all based on a palindrome of the left half of the lac diation Laboratory show that these crystals diffract to better operator with the base pair at the center of symmetry deleted than 3.5 A (data not shown). In addition, preliminary oscil- (40, 41). A 22-bp fragment of this sequence has been shown lation data have been collected by using a Nicolet area to bind the repressor 8-fold more tightly than the wild-type detector. Several hundred frames collected from a single operator sequence (41). The relative repressor tetramer- crystal had reasonable internal agreement for the symmetry to-operator DNA ratio was initially made 1:2 based on the related reflections, Rsym = 9.7% [Rsym = EhyhJil(h) - I(h)il/ potential for the protein to bind at two sites on the DNA 72hYi7iI(h)iI, where l(h) is the mean intensity of the symmetry (22-24). In practice, a slight excess beyond this 2-fold ratio is related reflections and I(h)i is ith measurement of reflection optimal. We have also added four molar equivalents of h]. A search for heavy atom derivatives has been initiated. 2-nitrophenyl 8-D-fucoside. This anti-inducer has been re- We have so far identified two compounds, thimerosal ported to bind to operator-bound repressor (42) and increase (C9HgHgNaO2S) and the sodium chloride salt of iridium the half-life of the complex with DNA. (Na3IrCI!6), that cause intensity changes from the native Cocrystals have been obtained with four of the DNA patterns in precession photographs. fragments. The 17-bp strand gave needles; the 21-mer gave lac repressor-IPTG complex. IPTG is a gratuitous inducer showers ofsmall football (NFL, USA)-shaped crystals.