Spiral structure of Escherichia coli HU␣␤ provides foundation for DNA supercoiling

Fusheng Guo and Sankar Adhya*

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-4264

Contributed by Sankar Adhya, January 4, 2007 (sent for review October 5, 2006)

We determined the crystal structure of the Escherichia coli nucleoid- Table 1. Statistics of data collection, molecular replacement, associated HU␣␤ protein by x-ray diffraction and observed that the and refinement heterodimers form multimers with octameric units in three potential arrangements, which may serve specialized roles in different DNA Diffraction datasets transaction reactions. It is of special importance that one of the Resolution, Å 2.5 structures forms spiral filaments with left-handed rotations. A neg- Unique reflection 7,730 atively superhelical DNA can be modeled to wrap around this left- Rmerge* (outer bin) 0.039 (0.399) handed HU␣␤ multimer. Whereas the wild-type HU generated neg- Compl., % (outer bin) 92.8 (72.9) ative DNA supercoiling in vitro, an engineered heterodimer with an Redundancy 6.2 altered amino acid residue critical for the formation of the left-handed Refinement spiral protein in the crystal was defective in the process, thus pro- No. of residues 167 viding the structural explanation for the classical property of HU to No. of nonhydrogen atoms 1,068 restrain negative supercoils in DNA. No. of solvents 24 No. of Ni 1 HU-multimers ͉ nucleoid ͉ x-ray crystallography No. of Cl 1 Resolution range, Å 20–2.5 R† (outer) 0.226 (0.357) he bacterial nucleoid is distinguished from the eukaryotic R † (outer) 0.258 (0.405) chromosome structure by the ostensible lack of an ordered free T rmsd bond, Å 0.017 nucleosomal structure, which characteristically influences gene rmsd bond , ° 1.514 expression . In Escherichia coli, the 4.6-Mb circular DNA rmsd chiral, ° 0.088 chromosome with a 1.6-mm circumference is condensed 1,000-fold to a diameter of 1 ␮m in the nucleoid (1). Although it has been *Rmerge ϭ Α͉I Ϫ͗I͉͘/ΑI, where I and ͗I͘ are the observed intensity and the mean suggested that DNA compaction in the nucleoid is caused by intensity of the same reflection from multiple measurements. Summation is extensive negative supercoiling, the involvement of several proteins, over all reflections. † ϭ Α ͉ ͉ Ϫ ͉ ͉ Α͉ ͉ e.g., HU, HNS, FIS, and SeqA, also in the compaction is now widely R ( Fo Fc )/ Fo , where Fo is the measured structure factor and Fc is the accepted (1, 2). However, the precise structure of the nucleoid, and calculated structure factor from the model. the exact role of these proteins in the nucleoid formation is unknown. Despite the deficiency in our knowledge about the three different forms, two of which exhibit distinct spiral multimers. nucleoid physical structure and any influence a structure may have The replacement of the ␤-E38 residue, critical for interdimeric on gene expression, it was found that specific amino acid changes interface in the left-handed spiral, by alanine affected the negative in the E. coli HU qualitatively change the morphology and the DNA supercoiling capacity in vitro. We discuss the significance of transcription profile of cells profoundly (3). The latter observation

the findings in nucleoid biology. BIOCHEMISTRY suggests that HU may be involved in nucleoid structure in an influential way not appreciated previously. Results HU contains two homologous subunits, ␣ and ␤, 9.5 kDa each. X-Ray Structure. The diffraction data were collected from a crystal HU also exists as ␣␣ and ␤␤ homodimers, in different stages of cell with a tetragonal space group I4 with unit cell dimensions of a ϭ growth (4). Although HU binds to DNA nonspecifically, it shows 1 b ϭ 82.915 Å and c ϭ 61.048 Å with one heterodimer in an a propensity for binding to distorted, bent, or Holliday-junction asymmetric unit. The parameters of data collection and refinement DNA, an ability that may aid DNA compaction (5). HU and are presented in Table 1. The local environments of the residues 12 topoisomerase I acting in concert also regulate DNA supercoiling ␣␤ both in vivo and in vitro (6, 7). In HU-deficient mutants, partially and 13 support the assignment of the heterodimers. In the ␣-subunit, E12 and K13 form two salt bridges with ␤-K18 of an relaxed DNA is compensated by altered levels of topoisomerase I ␤ or DNA gyrase (8, 9). Indeed, E. coli nucleoids in the absence of adjacent dimer and -E34 of the same dimer, respectively (Fig. 1a). In the ␤-subunit, A12 and G13 have no crystal contact (Fig. 1b). HU are significantly enlarged in volume (10). Ϫ ␣ ␣ That specific mutants of HU protein qualitatively change the cell The 2Fo Fc omit map of -E12 and -K13 shows apparent transcription profile suggests that the HU-mediated DNA struc- electron density at the site of the deleted residues at cutoff of 1.0 Ϫ tural change results in a defined architecture (topological or oth- and the corresponding Fo Fc omit map shows positive electron erwise) that guides the transcription pattern in cell. Although HU also plays specific roles in many DNA metabolic reactions (11–14), Author contributions: F.G. and S.A. designed research; F.G. performed research; F.G. and by well studied mechanisms, a relation between an HU-influenced S.A. analyzed data; and F.G. and S.A. wrote the paper. nucleoid structure and global gene expression profile is elusive. It The authors declare no conflict of interest. was also suggested that HU antagonizes, not aids, DNA supercoil- Data deposition: The structure factors have been deposited in the Protein Data Bank, ing (15). www.pdb.org (PDB ID code 2O97). ␣␤ We report here the structure of the E. coli HU heterodimer *To whom correspondence should be addressed at: National Cancer Institute, 37 Convent crystals determined by x-ray diffraction. The crystal structure of the Drive, Room 5138, Bethesda, MD 20892-4264. E-mail: sadhya@.nih.gov. heterodimer, unlike the structures of HU homodimers from E. coli This article contains supporting information online at www.pnas.org/cgi/content/full/ and other bacteria, show stacked dimers with octameric repeats in 0611686104/DC1.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611686104 PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4309–4314 Downloaded by guest on October 1, 2021 Fig. 2. Residues that contribute to the hydrophobic core of E. coli HU. Those in red directly contribute to dimerization. The underlined residues are differ- ent in ␣ and ␤ chains. The italicized residues are those whose counterparts in the opposite chain are polar or charged.

␣-K18, ␣-K73, ␤-K9, ␤-D15, ␤-K18, ␤-K75, and ␤-K83 are invisible. Although required for crystallization, the DNA fragment was missing in the electron density map. The crystals when dissolved in buffer showed a spectrum with significant OD at 260 nm, whereas pure HU␣␤ solution had no distinct absorbance at that wavelength. It is possible that a trace amount of DNase activity, intrinsic or extraneous, in the HU preparation damaged the DNA so as not to show any ordered electron density. Indeed, we detected a very low DNase activity in the preparation when a 32P-labeled pBR332 plasmid DNA was incubated overnight at 37°C with the HU preparation supplemented with 10 mM MgCl2. Because different DNA duplexes (SI Table 2) used in crystallization enabled HU␣␤ to form the same type of crystals, perhaps DNA molecules acted as a ‘‘general salt’’ in crystallization.

General Structure. The secondary structural elements of the HU␣␤ heterodimer are comparable to the previously reported structures of HU homodimers of Thermotoga maritima, Bacillus stearother- mophilus, Anabaena, and HU␣␣ of E. coli (16–20). The superim- position of the structures of the ␣- and the ␤-subunits gives an rms deviation (rmsd) of 0.895 Å per 69 C␣ atoms. The structures of the E. coli HU␣␤ heterodimer reported here and that of the ␣␣ homodimer (16) are superimposable with an rmsd of 0.807 Å per 140 C␣ atoms. The superimposition of two ␣␤ heterodimers, when the ␣- and ␤-subunits of one dimer are reversed, gave an rmsd of 0.905 Å per 139 C␣ atoms. Both ␣ and ␤ chains in the heterodimer consist of three helices and three antiparallel strands: helix 1 (residues 2–13), helix 2 (17–38), helix 3 (82–90), strand 1 (40–45), strand 2 (47–54), and strand 3 (75–81). The B-factor profiles indicate that the ␤-subunit residues, par- ticularly the first 20 and the last 10, are slightly less ordered than the ones in the ␣-subunit in the crystal packing environment. The folding of a monomer is maintained by core hydrophobic residues; Fig. 1. Snapshots of electron density maps of residues 12 and 13 as finger residues L6, I7, I10, A11, A21, and L25 interact with each other to prints of the ␣ and ␤ chains. (a) E12 and K13 of the ␣-subunit. (b) A12 and G13 generate a V- between helix 1 and helix 2, whereas residues ␤ Ϫ of .(c) The same region of a with E12 and K13 omitted. The 2Fo Fc map ␣-I32 (␤-V32), L36, V42, F50, V52, and P77 hold helix 2 and the Ϫ cutoff at 1 is in green. The positive Fo Fc map cutoff at 3 is in red. The red and three strands in a given conformation in both subunits. Most of blue text indicates separate HU dimer molecules. these residues also participate in dimerization (see below), making the formation of monomers and dimers interdependent.

density at cutoff of 3.0, especially on the location of the side chain Dimerization. The two monomers interwind into a dimer as a unit. ␣ ⌲ of - 13 (Fig. 1c). The Ramachandran plot shows that 92.6% of As reported in the HU homodimers (16–20), the heterodimers also residues fall in the most favored region, 5.8% fall in the additional hold extensive intersubunit hydrophobic interactions. The residues allowed region, and 1.7% fall in the generously allowed region that contribute to the hydrophobic core environment are listed in [supporting information (SI) Fig. 8]. Fig. 2. However, in contrasts to the symmetric intersubunit con- The assignment of the Ni- and a Cl-ions was as follows. A very figuration of the E. coli ␣␣ homodimer (16), the intersubunit massive electron density was found that does not fit with any amino interactions of the ␣␤ heterodimer reported here are asymmetric acid residues and other molecules in the mother liquor except Ni- (SI Fig. 9). The details of the dimerization interface are provided and Cl-ions. Detectable anomalous signals for heavier atoms were in SI Materials and Methods. observed in this spot. Residues 55–74 of ␣-chain and 56–73 of ␤ that The sequence difference and the resulting asymmetric dimeric are known as DNA binding arms, as well as the residue I16 located interactions, but not the basic folding, are driving forces for the in the turn between helix 1 and helix 2 in ␤, and the side chains of multimeric arrangement of HU␣␤ heterodimers, and thus the

4310 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611686104 Guo and Adhya Downloaded by guest on October 1, 2021 BIOCHEMISTRY

Fig. 3. Top views of crystal packing: an octameric structure with three possible arrangements. (a) The crystal packing lattice. (b) Arrangement I octamer. (c) Arrangement II octameric unit of a right-handed spiral multimer. (d) Arrangement III octameric unit of a left-handed spiral multimer. Red and blue represent the ␣- and ␤-subunits, respectively.

potential for the formation of higher-ordered nucleoprotein com- (684.9 Å2) and shows extensive interactions mainly between two plexes as described below. adjacent ␣-subunits (Fig. 4b). First, a Ni-ion, identified by RefMac5 software, participates in the interdimer interactions at this interface HU Multimers and Interdimeric Interfaces. The crystal packing data by forming coordination bonds with the ␣-M1 amide group, the showed that the heterodimer forms oligomers in three possible ␤-D40 side chain, the ␣-H54 side chain of the adjacent ␣-subunit, arrangements, I, II, and III (Fig. 3). A total of three interdimeric and three water molecules. Two of the water molecules are H- interfaces (a, b, and c) exist in the higher oligomers (Fig. 4). In bonded with a Cl-ion on the opposite side of the Ni-ion. The ␣-Q5 interface ‘‘a,’’ which has the buried solvent-accessible surface of side chain also participates in the Ni H-bond network. Thus, the 566.1 Å2, van der Waals interactions are involved between residues Ni-ion participates both in monomer–monomer and dimer–dimer 15–19 (E, L, S, K, and T) in-between helix 1 and helix 2 of an interactions. In addition, ␣-K3 forms a salt-bridge with ␣-E38 of the ␣-subunit with the same in an adjacent but symmetrically inverted adjacent dimer. The ␣-T4 forms three H-bonds with the adjacent ␣-subunit in an antiparallel mode (Fig. 4a). The side chains of dimer; the ␣-T4 amide and side chain form H-bonds with the ␣-E38 ␣-E12 and ␣-E15 interact with the side chains of ␣-K18 and ␣-T19, carbonyl, whereas the ␣-T4 side chain forms an H-bond with the respectively. This interface is present in arrangement I only (Fig. 3 ␣-D40 amide. Interface ‘‘b’’ is present in both arrangements I and a and b). Interestingly, the residue I16 located in the turn between II (Fig. 3 a and c). helices1and2in␤ is invisible in our heterodimer structure The buried solvent accessible surface of interface ‘‘c’’ is 290.1 Å2. indicating that the turn may potentially interact with another Interface ‘‘c’’ has two H-bonds between the ␤-E38 side chain of one protein. dimer and the two amide groups between ␣-G46 and ␣-K83 of Interface ‘‘b’’ has the largest buried solvent accessible surface another adjacent dimer (Fig. 4c). ␤-E38 and ␤-G39 make a turn

Guo and Adhya PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4311 Downloaded by guest on October 1, 2021 Fig. 5. Stereoviews of modeled HU␣␤ octameric unit of the left-handed multimer with DNA fragment as a repeating unit of a spiral structure. The ␣-subunit is in red, and the ␤-subunit is in blue. (a) Top view. (b) Side view. The HU␣␤ multimeric spiral is wrapped by DNA in a left-handed solenoidal structure.

As mentioned, we observed three potential multimeric arrange- ments (I, II, and III) in the crystal lattice. In I (Fig. 3b),twoofthe ␣␤ dimers interact with each other by using interface ‘‘a’’ to form a planar tetramer with the ␤-hairpin arms of each dimer available for interaction with DNA. By using interfaces ‘‘b’’ and ‘‘c,’’ this tetramer is stacked on another planar tetramer, in which each ␣␤ heterodimer is arranged in an inverted fashion such that the two DNA binding domains are interior. The oligomerization in arrangement II (Fig. 3c) uses interface Fig. 4. Diagrams of the interdimeric interfaces of E. coli HU␣␤ protein. (a) ‘‘b.’’ In this, each dimer is stacked on another to make an indefinite Interface ‘‘a’’ involves interactions between residues (E12 and E15-L16-S17-K18- spiral staircase. It takes four heterodimers to make a 360° rotation T19) of two adjacent -related ␣-subunits located in the turn between helix 1 and helix 2. (b) Interface ‘‘b’’ shows extensive interactions involving a of the spiral, which is right-handed. Interestingly, arrangement III, Ni-ion, a Cl-ion, three H2O molecules, ␣-M1, ␣-K3, ␣-T4, ␣-Q5, and ␣-D40 from one which uses interface ‘‘c’’ for oligomerization, also generates a dimer, and ␣-E38, ␣-H54, and ␤-D40 from the adjacent dimer. (c) Interface ‘‘c’’ continuous spiral with four stacked heterodimers making a full involves the side chain of the ␤-E38, which rotates to form two H-bonds with the rotation except that the spiral is left-handed (Fig. 3d). In both backbone amines of ␣-G46 and ␣-K83, respectively, from the interacting adjacent arrangements II and III the DNA binding domains in each dimer dimer. Red and blue represent segments of the subunits of ␣ and ␤, respectively. is projected outward from the spiral-body. In all three cases, interdimeric bonds, and so interdimeric surfaces are limited. Thus oligomers may not form in solution. In fact, HU is known to exist between helix 2 and strand 1 that serves as a turning point interwinding the monomers in dimers. The ␣-G46 residue is located as dimers in solution (21, 22). in the hydrophobic ␤-turn between strands 1 and 2, and belongs to the conserved motif (G-F-G) at region 46–48. The residue ␣-K83 Model of HU–DNA Complex. Because crystal packing arrangements is a transition point between strand 3 and helix 3. All of the residues II and III can generate right- and left-handed spirals of stacked that participate in the interdimer interactions in interface ‘‘c’’ are heterodimers (Fig. 3 c and d), the two configurations were modeled conserved (except E38) in HU family and located on turns. The with DNA by using Protein Data Bank entry 1P71 coordinates of NMR studies of B. stearothermophilus HU showed that the residues the Anabaena HU–DNA complex (20). The HU␣␤ dimer-DNA G39, F47, and G82 possess higher flexibility in solution; the higher model is shown in SI Fig. 10. In the two DNA–HU(␣␤) complexes, flexibility of the turn may allow participation in protein-protein the right-handed spiral could not accommodate a continuous piece interactions (19). The interface ‘‘c’’ is present in arrangements I and of DNA for interaction with each DNA binding loop in an octamer III (Fig. 3 a and d). unit in modeling. However, the left-handed spiral was modeled with

4312 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611686104 Guo and Adhya Downloaded by guest on October 1, 2021 Fig. 7. EMSA of HU-DNA. Five nanomolar relaxed plasmid samples were treated with wild-type ␣ϩ␤ϩ (a) or mutant ␣ϩ␤-E38A (b) HU at 0, 1.25, 1.39, 1.56, 1.79, 2.08, 2.50, 3.13, 4.17, 6.25, 12.5, 25.0, and 50 ␮M concentrations (lanes 1–13, respectively).

heterodimer allows the subunits to bind DNA differently compared with the way the homodimers bind and provide a structural basis for binding of DNA to multimeric HU. Although HU exists as dimers Fig. 6. Two-dimensional agarose gel electrophoretic of circular DNA in solution, it does form oligomers under certain conditions (6, 24). plasmids. Five nanomolar relaxed plasmid samples were treated with wild-type The oligomers may also form when bound to DNA. Consistently, ␣␤ ␣ϩ␤ ␮ HU (a) and HU -E38A (b) at 0, 1.79, 2.08, 2.50, 3.13, 4.17, and 6.25 M HU binding to DNA is cooperative (24, 25). Cooperativity is usually concentrations (lanes 1–7, respectively) in the presence of topoisomerase I (see text). the result of interactions between adjacent DNA binding protein molecules. We believe that HU uses interface ‘‘c’’ defined above for cooperative binding. DNA in an interpretable manner (Fig. 5 a and b). We assumed that The physiological relevance of the multimers needs further the E. coli heterodimer and the Anabaena homodimer share the consideration. The multimer with arrangement I may be involved same DNA binding site and a similar binding mode. In this model, in specialized functions like playing an architectural role in DNA– each dimer covers 16–17 bp with Ϯ1 nt uncertainty caused by one multiprotein complexes involving DNA looping, Holliday-junction base flipping-out in each strand in the Anabaena complex. It left formation, etc. (26, 27). We also speculate that arrangement II may one DNA bend angle in each heterodimer–DNA complex identical participate in retaining positive DNA supercoils locally in the to one of the two bend in the Anabaena complex. However, chromosome under some conditions. Our modeling shows that a it was not clear which monomer (␣ or ␤) is associated with the bend DNA can wrap around the multimer with arrangement III with angle. Four HU␣␤ dimers form one turn in the spiral, which is negative superhelicity. In this model, the asymmetry in the basic wrapped by a 64- or 68-bp DNA segment in the spiral groove. heterodimeric structure ensures negative DNA superhelicity when the oligomers of arrangement III bind to a continuous DNA. An HU octamer (a tetramer of the dimers) is the basic repeating unit DNA Supercoiling by HU. The left-handed ␣␤ spiral-DNA model around which a 64- or 68-bp-long DNA helix makes contacts with provides the structural explanation of the previously reported the protein creating one toroidal DNA with negative superhelicity. finding that HU restrains negative supercoils in DNA (1). To test BIOCHEMISTRY This potential of HU is consistent with the following observations: the idea that structure III generates negative supercoils, a mutant (i) HU is a major nucleoid protein (28); (ii) HU forms nucleosome- HU␣␤ was made in which E38 in ␤ was changed to an alanine like structures (29, 30); (iii) HU binds to DNA cooperatively (24, disrupting a critical interaction at interface ‘‘c.’’ We tested the DNA 25), presumably by using the interfaces of arrangement III; (iv)HU supercoiling activity of the wild-type HU␣␤ and the altered ␣ϩ␤ could form cylindrical structures around which DNA is wound in a HU -E38A as described in SI Materials and Methods (Fig. 6). toroidal supercoils (6); (v) HU increases the thickness of DNA from Although the results confirmed the ability of wild-type HU to 20 to 60 Å (7); (vi) plasmid DNA extracted from HU-deficient cells generate and retain negative superhelicity in DNA when the DNA are partially relaxed (31); (vii) HU makes negative supercoils in incubation with HU was followed by treatment with topoisomerase DNA in the presence of topoisomerase I in vitro (6); and (viii)a I (Fig. 6a), it is clear that the mutant HU protein was substantially mutant HU defective in generating the potential left-handed spiral defective in DNA supercoiling (Fig. 6b), suggesting an in vivo role observed in the crystal is inefficient in creating negative DNA of structure III. The wild-type and the mutant HU bind to DNA supercoils as reported here. In the left-handed HU spiral, the with comparable affinities by EMSA (Fig. 7). residue E38 of a ␤-subunit forms H-bonds with two backbone amide groups of the ␣-subunit of a neighboring dimer through Discussion interface ‘‘c’’ (Fig. 4c). This interaction is weak, which may allow The ␣- and ␤-chains of HU heterodimer in E. coli have 30% a dynamic balance between HU dimers and multimers depend- difference in amino acid sequence. The nonhomology is very likely ing on environments. Consistently, higher salt concentrations responsible for causing the asymmetric heterodimeric structure, abolish dimer–dimer interactions (6). Our experiments showing which is distinct from the more or less symmetric structure of the that the HU␣ϩ␤-E38A mutant showed significantly decreased homodimers in E. coli and other bacteria (16–20). Whether the ␣␣ ability in generating negative DNA supercoils compared with the and the ␤␤ homodimers in E. coli have any different from wild type strongly supports the idea that the left-handed spiral the heterodimer, other than the instability of the ␤␤ homodimer (structure III) is real and wraps DNA around it in generating (23), is not known. negative superhelicity. The crystal structure of the HU␣␤ heterodimer suggests that it From the results of DNase I digestion of DNA-bound HU, it was may form higher oligomers. The observed asymmetry in the shown previously that the nuclease cut sites are made with a

Guo and Adhya PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4313 Downloaded by guest on October 1, 2021 periodicity of 8.5 bp (6). Our modeling shows that a complete 1B8Z) (17) as a search model. The electron density map generated superhelical DNA turn around the left-handed HU spiral contains from the experimental absolute structure factor and the phase from Ϸ64 or 68 base pairs, i.e., Ϸ8 or 8.5 base pairs per HU␣␤ monomer. the molecular replacement output coordinates was improved to be This is in complete agreement with the results of DNase I diges- more traceable by the Density Modification (DM) program with a tion (6). solvent content of 42%. RefMac 5.0 was used for the rigid body, The left-handed superhelical DNA around a tetragonal HU restraints, and translation–libration–screw refinements. The elec- structure III giving rise to a 64- or 68-bp solenoidal structure is tron density maps were calculated with the FFFbig program. The reminiscent of eukaryotic nucleosome structures. Indeed, the ex- omit maps were generated by running the ‘‘refine.inp’’ and istence of nucleosome-like structures in bacterial chromosome has ‘‘model࿝map.inp’’ programs of crystallography and NMR system been predicted before (29, 30). However, the bacterial ‘‘nucleo- (36). The starting temperature was set at 2,000 K in the annealing some’’ structure deduced here from the multimeric arrangement III process. The solvent-accessible areas were calculated with the may differ from the eukaryotic counterpart. In the eukaryotic AREAIMOL program. The molecular model was visualized and nucleosome, each core unit restraining negative superhelical DNA manipulated with the O program (37). The graphs were generated is separated from a neighboring unit by spacer DNA (32), whereas with MolScript (38), Raster3D (39), and the PyMOL Molecular in the bacterial case, octameric HU units wrapped by negatively Graphics System (www.pymol.org). The superposition of the HU supercoiled DNA are stacked one on another as in a spiral staircase protein structure of the Anabaena HU homodimer–DNA complex (20) to the E. coli HU(␣␤) heterodimer brings the DNA structural without spacer DNA. Our results demonstrate that by providing a coordinates of the former to the corresponding region of the latter foundation for negative superhelicity in DNA, HU␣␤ can form a giving rise to an E. coli HU(␣␤) heterodimer–DNA complex (SI ‘‘nucleosome-like’’ structure, presumably regionally. This structure Materials and Methods). would help conversion of a plectonomic negatively supercoiled DNA circles into a more open ring with nucleosomal ‘‘bead’’ DNA Supercoiling Assays. The supercoiled circular plasmid pFLEX- distributed as was shown by atomic force microscopy (29). gap6 was relaxed by E. coli topoisomerase I. One hundred- Materials and Methods nanogram relaxed DNA samples were incubated at 37°C for 30 min with 0.036–0.50 ␮g of wild-type or mutant HU in 10-␮l solutions Wild-type and mutant HU proteins were purified from an expres- containing 35 mM Tris⅐HCl (pH 8.0), 20 mM NaCl, and 5 mM sion plasmid (pRLM118) donated by R. McMacken (33). The DTT. Calf thymus topoisomerase I (0.5 ␮l, 5–15 units/␮l; Invitro- ␣ϩ␤ HU E38A mutant was made as described in the SI Materials and gen) was added, and incubation continued for 2 h. Ten micrograms Methods. The purified proteins were concentrated by Amicon of proteinase K was then added and incubated for another 30 min. Centrifugal Filters (Millipore, Billerica, MA) to 16.4 mg/ml in a The DNA samples were analyzed by two-dimensional agarose gel solution containing Tris⅐HCl (20 mM, pH 8.0), KCl (0.5 M), and electrophoresis in a 4.5 ϫ 4.5-square-inch gel. The first-dimension glycerol (5%). Protein concentration was measured by using the electrophoresis was run at 0.25ϫ TBE for Ϸ15 h, and the second- BCA Protein Assay Kit (Pierce, Rockford, IL) with BSA as dimension electrophoresis was run at 0.4ϫ TBE containing 1 ␮g/ml standard. HPLC-purified DNA oligomers (SI Table 2) from Sigma- chloroquine for 3 h. The DNA binding assays were as before (40). Genosys (The Woodlands, TX) were dissolved in pure water to a One hundred-nanogram relaxed plasmid samples were incubated at concentration of 10 mM. Crystallization reagents were from Hamp- 37°C for 30 min with 0.025–1.0 ␮g of wild-type or mutant HU in a ton Research (Aliso Viejo, CA). Details of crystallization are 10-␮l solution containing 35 mM Tris⅐HCl (pH 8.0), 20 mM NaCl, provided in SI Materials and Methods. The frozen crystals were used and 5 mm DTT. The DNA samples were analyzed by agarose for x-ray diffraction by an in-house x-ray diffractometer with a electrophoresis in 4.5 ϫ 4.5-square-inch gels in 0.25ϫ TBEfor15h. Mar345 imaging plate detector and a Rigaku RU-3H x-ray gener- ator. Data were collected at 1° rotation for each frame and were We thank our colleagues for assistance, especially S. Kar for her work in HU reduced by the Denzo program and scaled by the Scalepack that prompted us to determine the structure, and T. Soares for purification program of the HKL package (34). The CCP4 Interface was used of proteins. We are grateful to D. Xia, F. Dyda, V. Zhurkin, M. Tolstorukov, P. Rice, and D. Davies for critical reading of the manuscript and helpful in molecular replacement and model refinement (35). A 5% suggestions. The work was supported by the Intramural Research Program FreeR-flag was inserted in data reduction. The molecular replace- of the National Institutes of Health, National Cancer Institute, Center for ment was conducted with the Molrep program by using the dimeric Cancer Research. We used the high-performance computational capabil- Thermotoga maritima HU coordinates (Protein Data Bank entry ities of the Helix Systems at the National Institutes of Health.

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