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Intermolecular Contacts Between the λ-CroRepressor and the Operator DNA Characterized byNuclear Magnetic Resonance SpectroscopyHidehito Tochio a , Chojiro Kojima a , Hiroshi Matsuo a , Toshio Yamazaki a &Yoshimasa Kyogoku aa Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka ,565-0871 , JapanPublished online: 21 May 2012.

To cite this article: Hidehito Tochio , Chojiro Kojima , Hiroshi Matsuo , Toshio Yamazaki & Yoshimasa Kyogoku(1999) Intermolecular Contacts Between the λ-Cro Repressor and the Operator DNA Characterized by NuclearMagnetic Resonance Spectroscopy, Journal of Biomolecular Structure and Dynamics, 16:5, 989-1002, DOI:10.1080/07391102.1999.10508309

To link to this article: http://dx.doi.org/10.1080/07391102.1999.10508309

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Intermolecular Contacts Between the A.-Cro Repressor and the Operator DNA Characterized by Nuclear

Magnetic Resonance Spectroscopy

http://www.albany.edu/chemistry/sanna/jbsd.html

Abstract

The specific interaction between A. phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13Cf15N- labeled A.-Cro in complex with unlabeled DNA, chemical shift assignments of the A.-Cro-DNA complex were obtained using heteronuclear NMR experi­ments. Inter-molecular contacts between the protein and DNA were identified using het­eronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOB constraints to dock A.-Cro to the bent 8-form DNA using restrained molecular dynamics. The structure of one of the subunits of A.-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecu­lar NOEs were observed between the "helix-tum-helix" region comprising the a2 and a3 helices of the A.-Cro protein and the major groove of the DNA. The methyl group of Thrl7 forms a hydrophobic contact with the methyl group of the thymine at base pair I in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of A.-Cro to several variants of the operator sequence.

Introduction

The repressor protein A.-Cro binds to right operator (OR) regions in the A. phage genome in competition with the ci repressor (1). A.-Cro consists of 66 amino acids and fonns a dimer in solution. The crystal structure of A.-Cro (2) inspired a model of the interaction between A.-Cro and an operator DNA (3). In this model, the a3 helical region (Tyr26 toArg38) ofA.-Cro binds to the major groove ofB-fonn oper­ator DNA and is primarily responsible for recognition. This complex has been well characterized using protection from methylation, site directed mutagenesis of both the DNA bases and protein amino acids in addition to thennodynamic and NMR studies (4-12). Structural distortions at the center of the OR3 fragment of DNA upon binding have been identified using NMR (13). Furthennore, Tyr26 and His35 were found to interact with the OR3 DNA fragment using photo CIDNP (14). Changes in the proton chemical shifts of Tyr51, Ile40 and Leu42 upon binding sug­gested that the structural changes occurred at the inter- and intra-subunit ~-sheet regions of the A.-Cro protein (15,16).

A refined crystal structure (3.0A) of A.-Cro in complex with consensus operator DNA revealed that the a3 helix is located within the major groove of the DNA ( 17, 18). Furthennore, large global confonnational changes in both A.-Cro and the operator DNA were observed upon binding. One monomer of the A.-Cro dimer rotates by about 53° relative to the other. This confonnational change is facilitat­ed by a twisting of the ~-strands connecting the two subunits. Similarly, the oper­ator DNA was bent by about 40° into the shape of a boomerang but maintained the

Journal of Biomolecular Structure & Dynamics. ISSN 0739-J/02 Volume 16, Issue Number 5, (1999) ©Adenine Press (1999)

Hidehito Tochio, Chojiro Kojima, Hiroshi Matsuo, Toshio Yamazaki and Yoshimasa Kyogoku* Institute for Protein Research,

Osaka University,

3-2 Yamadaoka,

Suita, Osaka 565-0871, Japan

*Author to whom correspondence should be addressed. Phone: 81-6-6879-8598; Fax: 81-6-6879-8599; E-mail: kyogoku@protein.osaka­u.ac.jp

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Figure 1: Sequences of the operator DNA oligomers that the A.-Cro protein specifically binds. Dissociate constants (K0 ) are indicated for 21-mer (Kim et al., 1987)and 17-mer(Torigoeetal.,I991).Conservation of sequence of the SYMG 17-mer is shaded.

Watson-Crick B-fonn. The crystal structure revealed detail of contacts between the A.-Cro and the DNA fragment, which made it possible to explain discrimination of the OR3 from other right operators (17,18).

On the other hand, to elucidate the molecular details of operator recognition in solution, the structure of A.-Cro was determined using NMR ( 19). This study has been extended to the complex of A.-Cro and operator DNA. Right operator (OR) region of A. phage genome consists of three regions including OR I, OR2, and OR3. A.-Cro binds most strongly to OR3 and most weakly to OR2 (20). However, the A.­Cro-DNA complex using the OR3 17-mer is not so stable but a complex using a 17-mer DNA containing a consensus operator sequence is more stable. This con­sensus sequence is conserved among all operator sequences that are bound by A.­Cro. We employed the 17-mer DNA with symmetric consensus sequence charac­terized by a G-G mismatch at the center of the duplex (SYMG 17-mer, Figure 1). A.-Cro has about 10 times higher affinity for the SYMG 17-mer than for OR3 17-mer. The dissociation constants (K0 ) of the A.-Cro-DNAcomplex with OR3 17-mer and SYMG 17-mer were 1.30 x 10-6 and 1.58 x 1Q-7, respectively (21). In addi­tion to producing a stable complex, the symmetry of SYMG 17-mer greatly sim­plified the NMR spectra as A.-Cro bound to the DNA as a symmetric homodimer.

We prepared both I5N- and 13Cfi5N- labeled A.-Cro in complex with unlabeled SYMG 17-mer. The NMR chemical shifts were assigned using heteronuclear NMR techniques, and intermolecular NOEs were assigned using filtered NOESY experiments. The inter-molecular NOEs were used to produce a complex model of the A.-Cro and the SYMG 17-mer DNA fragment using restrained molecular dynamics. The intermolecular NOEs indicated the role of methyl groups of thymine to distinguish the OR3 from other OR operators.

Materials and Methods

Preparation of A.-Cro-DNA Complex

A.-Cro was derived from transformed E. coli (strain TG1) cells containing the expression plasmid, pKH1500. In order to achieve complete I5N and/or I3C label­ing of the protein, the bacteria were grown in M9 minimal medium with 15NH4Cl and/or 13C-labeled glucose as the sole source of nitrogen and/or carbon, respec­tively. The A.-Cro protein was purified as described previously (22,23).

The SYMG 17-mer DNA was not labeled. The SYMG 17-mer, containing a trityl (diemethoxytryphenylmethyl) residue at the 5'-end, was purchased from Vector Research ltd. The oligomer was loaded onto a reverse phase cosmosil C18 HPLC column equilibrated with 5 % acetonitrile in water containing 0.1 M triethylam­moniumacetate and then eluted using a linear acetonitrile gradient. After removing

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the trityl groups with 80% acetic acid, HPLC using a Cl8 column was repeated. The counter cations (triethylammonium ion) bound to the DNA oligomers were exchanged to sodium by anion exchange FPLC using a Mono-Q packed column. Gel filtration HPLC in water using a G3000 PW packed column was used to remove the unbound salt.

Each A.-Cro-DNA sample contained 2.3 mM A.-Cro protein as monomer and 1.3 mM DNA (about 1.1 equivalent) in 20 mM phosphorous buffer (pH 6.8) contain­ing 50 mM KCl and 1 mM EDTA in either 99.996% 2H20 or 90% H20110% 2H20.

NMR Spectroscopy

NMR measurements were performed on a Bruker DMX500 spectrometer equipped with a triple resonance IH (ISN, 13C) probe for IH detection. All spectra were recorded at 35-45°C. The spectra were processed and analyzed on Silicon Graphics Iris Indy workstations using NMRPipe and NMRPipp (24,25).

3D CT-HN(CO)CA and CT-HNCA (26) spectra were acquired to assign the back­bone atoms of the A.-Cro in the complexed form. The spectral width of the I3Ca, ISN and IH dimensions were 6915.63, 1008.06 and 12500 Hz, respectively. Quadrature detection in tl and t2 dimensions was achieved using the TPPI-States method (27). The number of complex points acquired was 46 in the indirect car­bon, 22 in the indirect nitrogen, and 512 in the direct proton dimension. A 3D IH­ISN-IH NOESY-HSQC was acquired to obtain intra-molecular NOE connectivities for the A.-Cro protein. A modified pulse sequence proposed by Marion et al. (28,29) and Zuiderweg et al. (30) was used with a 100 msec mixing time. Solvent sup­pression was achieved using WATERGATE sequence (31). The spectral width of the indirect proton, nitrogen and acquisition dimensions were 6500, 1250 and 5000 Hz, respectively and the sizes of the acquired data matrix were 128 (I H)* 16 (ISN) * 512 (IH) complex pairs. ISN decoupling during acquisition was achieved using WALTZ-16 (32). A 3D IH_I3C_IH HSQC-NOESY spectrum was acquired with an NOE mixing time of 100 ms. The spectral widths of the indirect proton, carbon and direct dimensions were 5000, 4000 and 5000 Hz, respectively while the number of complex points acquired was 128, 32, and 512, respectively. 13C decoupling during acquisition was done using GARP-1 (33). A 3D HCCH-TOCSY (34,35) spectrum was acquired as described by Kay et al. (35) except that 180° composite pulses were used to compensate for imperfect 180° pulses instead of gradient pulses. The DIPSI-3 isotropic mixing scheme was used with a 16 msec mixing time. The spec­tral widths of the tl (IH), t2 (13C) and t3 (IH) dimensions were 3500.92,4464 and 6009.615 Hz respectively, and the number of complex points acquired were 128 (tl), 32 (t2), and 512 (t3). 13C decoupling during acquisition was achieved using the GARP-1 phase modulation (33).

Assignments of non-labile IH resonances of the complexed SYMG 17-mer were obtained using I3C filtered (fl, f2) 2D IH-IH NOESY and I3C filtered ~fl. f2) 2D IH-IH COSY experiments (36,37). An NOE mixing time of 100 msec was used with spectral widths 5000Hz in tl and 6250Hz in t2 and with 256 and 512 com­plex points in the indirect and direct dimensions, respectively. 13C decoupling dur­ing acquisition was done using the GARP-1 phase modulation (33). ISN filtered (fl, f2) 2D IH-IH NOESY and 2D IH-IH NOESY spectra were used to assign the exchangeable protons. Spectral widths of9500 and 10000 Hz were used in the indi­rect and acquisition dimension, respectively with 256 (tl) and 1024 (t2) complex points. ISN decoupling during acquisition was achieved using WALTZ-16 phase modulation (32) and an NOE mixing time of 100 msec.

To obtain inter-molecular NOEs between A.-Cro and the SYMG 17-mer, both 13C selected (fl) 13C filtered (f2) 2D IH-IH NOESY (36,37) and 13C filtered (f3) 3D 1H-13C_IH HMQC-NOESY spectra were acquired. The I3C filtered (f3) 3D IH_I3C-

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Table I Inter-molecular NOEs between A.-Cro and the DNA 17-mer.

A.-Cro protein SYMG 17-mer Thr 17 y-methyl Tl H5-methyl LysiS E-H Tl H5-methyl Val25 y-methyl Tl2 HI'

Tl3 H5-methyl Tl3 H6 Tl3 HI' Tl3 H3'

Tyr26 E-H Tl3 HI' Gl4 H3'

Ala29 a-H Tl3 H5-methyl Ala29 ~-methyl GI2 HI'

Tl3 H5-methyl Tl3 H6 Tl3 HI' Tl3 H3'

IH HMQC-NOESY spectrum was measured on a JEOL Alpha 600 spectrometer equipped with a pulsed field gradient unit using the pulse sequence proposed by Lee et al. (38). A NOE mixing time of 100 ms and spectral widths of 6002.4, 6000.24 and S99S.2 Hz in the indirect proton, indirect carbon, and direct dimension were used, respectively. Furthermore, 128, 16, and S12 complex points were acquired in the indirect proton, indirect carbon, and direct dimension, respectively.

To identify the secondary structural units of the A.-Cro protein, amide HID exchange rates were determined. The A.-Cro-SYMG 17-mer complex was lyophilized and then dissolved in 2H20. IH- 15N HSQC spectra (39,40) were record­ed after 20 min, 1hr, and 11 hr of dissolving into 2H20 at 3Scc.

Constraints Used to Generate the Model of the Complex

Distance constraints were obtained using 2D and 3D NOESY spectra. Intra-mole­cular distance constraints were classified as either strong, medium, or weak based on peak height and used to set upper proton-proton distances of 3.S, 4.0 and S.OA, respectively. The methyl and methylene protons were treated as pseudo atoms and corrections of 1A were added to constraints including these atoms. For all observed inter-molecular NOEs, S.OA was assumed as the upper limit for proton-proton dis­tance. A lower limit of 1.8A was used for all NOE derived distance constraints. Slowly exchanging amide protons within secondary structural elements were assumed to be involved in hydrogen bonds and were included in the constraint list by using an upper limit of 3.0A for the N-0 distance and 2.0A for the HN-O dis­tance. Furthermore, the presence of imino signals was used to indicate hydrogen bonding within the DNA. Consequently, hydrogen bond constraints were included for all base pair except the mismatched base 9Gua to maintain base pairing in the DNA. The constraints used were summarized in Table II.

Model Building

The structure of the A.-Cro protein in the complex form was calculated by the simu­lated annealing method (41) using the intra-protein constraints on Silicon Graphics workstations with the X-PLOR version 3.1 ( 42). The NMR structure of the DNA free A.-Cro ( 19) was employed as an initial structure. The initial structure was exposed to 7 .S psec of simulated annealing at high temperature ( 1000 K) followed by gradual cooling to 100 K for 3.7 psec and then minimization of the target function.

The structures of A.-Cro which satisfied all of the intra-molecular constraints and an ideal B-type DNA structure of the SYMG 17-mer sequence were used as the initial structure of docking calculations. The A.-Cro was initially positioned 1SA apart from the DNA in the orientation which satisfied the inter-molecular NOEs (Table I) on a CRT monitor using the molecular viewing software Midas Plus (43).

Docking calculations between the A.-Cro and the DNA fragment were conducted using X-PLOR on the initial structures. In this stage, all intra- and inter-molecular NOEs derived distance constraints and hydrogen bond constraints were used. Furthermore, in order to maintain the DNA structure to bent B-form, the phosphate atoms were also restrained according to the low resolved crystal structure of the complex of the A.-Cro and the DNA fragment bearing pseudo consensus sequence (44) by means of a pseu­do potential. A pseudo potential for maintaining the base pair planarity within the DNA was included in the target function. Additionally, we employed 72 loose torsion angle restraints characteristic of both A- and B-DNA along the DNA backbone to pre­vent problems associated with local mirror images. The angle restraints used included a= 60c ±soc,~= 180c ±soc, y= 60c ± 3Sc, £ = 180c ±soc, and 1; = -8Sc ±soc.

After 7S psec at high temperature (1000 K), the system was cooled down gradual­ly to 100 K for 37 psec. The target function was subsequently minimized.

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Table II Structural statistics for the 20 calculated structures!.

Distance constraints Protein

NOE All Intra-residue Sequential (li-jl= 1) Short range ( l<li-jl~5) Long range (li-jl>5) Inter-subunit

Intra-subunit H-bond Inter-subunit H-bond

DNA NOEAll

Intra-residue Sequential inter-residue Inter-strand

H-bond Protein-DNA inter-molecule Dihedral angle constraints for DNA

Base pair planarity constraints for DNA Position restraints for phosphorous atoms of DNA 2

Ramachandran plot (residues 3-59 of A. -Cro protein)3

Most favorable region Additionally allowed region Generously allowed region Disallowed region

Average r.m.s deviations from experimental distance restraints (A) Average r.m.s deviations from idealized covalent geometry

Bonds (A) Angles (degree) lmpropers (degree)

Average r.m.s deviations of atomic coordinates of A.-Cro protein

Backbone heavy atoms, residues 3-59 (A) Heavy atoms, residues 3-59 (A)

monomer 0.65 1.20

1,905 783 416 354 352 40 76

8

364 186 164 164 80 30 72

16 32

58.2 35.0 4.9 1.9

0.078±0.001

0.008±0.0000 1 1.1±0.007 0.54±0.016

dimer 0.85 1.34

I None of these structures exhibited distance violations greater that 0.5A or dihedral angle violations greater than 5°. 2These constraints were used to maintain the DNA structure to bent B-form according to the low resolved crystal structure (44). 3The program PROCHECK_nmr (53) was used to assess the quality of the structures.

Results

NMR Assignments

NMR samples were prepared with both 15N- and l3Cfl5N- labeled 1..-Cro in com­plex with unlabeled DNA containing the symmetric consensus sequence (SYMG) 17-mer. For assignment of the lH, l3C, and 15N resonances of 1..-Cro in the com­plex, heteronuclear multidimensional NMR experiments were performed. 90% of the proton resonances of the polypeptide backbone were assigned. The sidechain ~­and y-protons were assigned for 80% and 65% of the residues, which contained these protons, respectively. Most of methyl groups were assigned except Ile34, Leu 7 and Ala 46, as they were not observed in the HCCHTOCSY spectrum. Aromatic protons of all three tyrosine, Phe 14 and His 35 were assigned while those of Phe 14 and Phe 58 were not assigned because they were not observed even in I3C HSQC spectrum.

The 1H resonances of the DNA in the complex were assigned using a 2D 13C fil­tered (fl, f2) NOESY experiment in D20 and a 2D 15N filtered (fl, f2) NOESY experiment in H20 (36,37). These heteronuclear-filtering techniques could remove the intense 1H signals of the 1..-Cro thereby enabling assignment of the DNA.

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Thy13-H6

Excessive line broadening occurred at central three base pairs (Gua8, Gua9, and Cytl 0) causes some signals to disappear. Consequently, non-exchangeable base protons and I' sugar protons were assigned for 12 and 13 residues, respectively, while the 2'/2" and 3' protons were assigned for 11 residues. Imino protons were assigned for all residues except Gua9.

Assignment of Inter-molecular NOEs Between the A.-Cro Protein and the SYMG 17-mer

Inter-molecular NOEs were identified from 2D 13C selected (fl), 13C filtered (f2) NOESY (36,37) and 3D 13C filtered (f3) HMQC-NOESY experiments (38) in D20. Because of the low sensitivity of the I3C filtered experiments, intermolecu­lar NOEs observed were predominantly strong NOEs involving methyl groups (Table 1). Figure 2 shows a region of the 2D 13C selected (fl) 13C filtered (f2) NOESY spectrum.

Thy13-Hl' Gua13-H3'

Thyl-5Me Thy13-5Me

• I

-----ftl------------------f---t-------li---.~---li+-CI------------~·J-----·----~. 1

' ' .. ' •.••. ' ... ........ Aill'Jill~ ' l~ I f. ~ ' ' laiil ' ; '· I I · 1

·····-············-··-------------------r·---·-------.,t.~•,•r··-·t·---~·11·-o------------ 3-----·- K18£H

Figure 2: A region of nc selected (fl ), IJC filtered (f2)

20 NOESY spectrum observed for the complex of the A.-Cro protein and the SYMG 17-mer. The 2.3 mM sample of the A.-Cro-SYMG complex was dissolved in I 00% 2HP buffer. The spectrum was recorded at 35°C and pH 6.8. The assignments are indicated by the indi­vidual atom names and residue numbers.

7 6 5 4 3 2

Hppm

It is apparent from the NOEs between the methyl groups of the thymine residues and Thrl7 (a2 helix), Val25 (turn region) andAla29 (a3 helix) that the helix-turn­helix region of A.-Cro is located in the major groove of the DNA (Table I). This feature is confirmed by other inter-molecular NOEs involving nearby residues and is consistent with the binding mode in the crystal structure ( 44,17, 18).

1H.2H Exchange Rates of the Amide Protons

Amide exchange was considerably slower in the complexed form versus the DNA free form. While almost all of the backbone amide protons of the free protein were exchanged to deuterium after 143 min of resuspension in 2H20 (45), many of the backbone amide protons of the complexed form located within regions of regular secondary structure remained after 11 hours (Figure 3).

Cro-DNA Complex Model with Bent DNA

To characterize the A.-Cro-DNA complex, we used the intermolecular distance con­straints as input for restrained molecular dynamics to dock the protein to SYMG 17-mer. Unfortunately, the disappearance of the signals from the central base pairs resulted in very few distance constraints for these bases. Consequently, the DNA tends to kink severely at the center during the docking calculation as the intermol­ecular distance constraints localize on both sides of the DNA fragment. It appeared to be difficult to determine the DNA structure by only using NMR data. Therefore, we made a complex model maintaining the DNA structure to be a bent B-form since the bent of DNA upon specific binding to the A.-Cro has been indicated using quite a few of different methods ( 13,44,46-48). Hence, the coordinates of the phos­phorous atoms were restrained to be identical to the bent B-form DNA in the crys­tal of the A.-Cro-pseudo consensus I 7-mer DNA fragment complex (44). From 90 initial structures, our docking calculation gave 35 docked models of the A.-Cro­DNA complex that satisfied all of the intra-protein and inter-molecular distance

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• In H20 ~

• ~

0

6 ~ I> • !)

1 hr. ·a-

~-· 0

G24G>G15 0 V25

F14· oD19

105

110

995

Contacts Between the J..-Cro Repressor

0 : <>.o .. ·= ~t c

l34 cL23 T43 o o K21 • 130

115 e and the Operator DNA ~------------------~------------0

~. tl. : <}

0 ()~ ,N..

··r • • • 0. • til

20min.

G24<> GIS

• V25 Ft4o .019

134 o L23 T43 0 • K21 ,..130 All •.• <0-·0 R13

Y26• rE53 M12 °F58 022 diP

A20 A33 L? YIO Q>11'Y51

,. E54 ... 0 • 140

K56<> vss A29 L42co 0 F41

10.0 9.0 8.0 7.0 6.0 Hppm

All C>E53 0Mr2 .. Rl3 • •FSS

022 A20~'L7 A33

YIO<> o-YSI E54o e • L420 K;

6"F41

vss A29

11 hr. G24 ...

• V25 o019

134 •L23 T43o .. 121 «130

A114C E53 oM12 9 FSS

022 A20-G<>" L7 A33

YIOo--<>YSI E54~ q

K56. VSS A29 L42<> • F41

10.0 9.0 8.0 7.0 Hppm

6.0

120 z

125

105

110

115 5 ~

120 z

125

constraints with no violation greater than 0.5A. Furthermore, no intra-DNA dis­tance constraint showed violations greater than 0.5A despite the overall shape of the DNA being constrained to the bent B-form of the crystal structure. A.-Cro docked to the bent SYMG-17 mer is represented in Figure 4, and a ribbon diagram of a representative structure is shown in Figure 5.

Discussion

Protein-DNA Contacts Derived from Inter-molecular NOEs

The binding mode of A.-Cro to the operator DNA bearing the consensus sequence was characterized using the intermolecular NOEs (Table I). Intermolecular NOEs between y-methyl group ofThr 17 and the methyl group ofThyl implied that there is a hydrophobic interaction. This interaction was also found in crystal structures in complex with different DNA fragments bearing pseudo consensus sequence (44) and consensus sequence (17,18). Furthermore, theCa ofThr17 has been indicated within 12A of parts of Thy 16 and Ade 15 in the specific operator complex using phenylazide-mediated photocrosslinking studies (49). Other hydrophobic contacts between A.-Cro and the operator DNA were found among methyl groups of Val 25, Ala29, and Thy13 (Figure 6). The methyl group ofThy13 has been shown to be in a hydrophobic pocket using I9F NMR studies (5). The hydrophobic contact between Ala29 and Thy 13 is also found in the crystal structure ( 17, 18).

Extensive studies have been performed to investigate the effects of base substitu­tions on operator DNA using filter-binding assays (6,50). These studies revealed at the first base pair, aT-A base pair was preferential to binding over aU-A base pair, while at the fifth base pair, an A-T base pair was preferred over an A-U base pair. The differences of binding free energy were 1.1 and 1.6 kcal/mol for the substitu­tions at first and fifth base pairs, respectively. Furthermore, substitutions to other base pairs (G-C, C-G and A-T or T-A) at position 1 or 5, respectively, also reduced affinities to the operator DNA bearing consensus sequence (10,6). These findings confirm the importance of the methyl-methyl contacts observed between the thymine residues at the first and fifth position in the consensus sequence and Val25, Ala29, or Thr 17 in A.-Cro.

Figure 3: Exchange pattern of amide protons with sol­vent deuterons monitored by a series of the 1H-15N HSQC spectra of the A.-Cro-SYMG complex. The A.­Cro-SYMG-17 mer complex was lyophilized and then dissolved in 2Hp. IH.ISN HSQC were recorded after 20 min. !hr. and II hr of dissolving into 2HP at 35°C.

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Operator Variants

Among native operator sequences, A.-Cro binds most strongly to the OR3 sequence. However, the DNA fragment with the symmetric consensus operator sequence (SYMG 17mer) has about 10 times higher affinity for A.-Cro than the OR3 17mer (21) (Figure 1 ). This preference can be attributed to the hydrophobic contacts men­tioned above. While the left halves of these two sequences are identical bearing the consensus sequence, there are differences in the right half (Figure 1 ). For the bases which have the most contact with A.-Cro, namely, the six base pairs (base pairl to base pair 6), the left and right halves of the OR3 17-mer sequence are almost sym­metrical. The one exception is at the fifth base pair that has aT-A in the consensus left half and G-C in the right half. Intermolecular NOEs indicated that the methyl group of Thy, at the fifth base pair, forms a hydrophobic contact with the methyl groups of Val25 and Ala29 in the SYMG 17mer. Substitution of this T-A to a G-C disrupts these hydrophobic interactions causing the A--Cro protein to bind less tight­ly to the OR3 17-mer native sequence than to the consensus sequence.

The substitution of theT-A base pair for an A-T base pair at position 5 in the right half of the OR 1 sequence removes the hydrophobic contact between Thymine and Val25 or Ala29, thereby reduces the affinity of A.-Cro for OR 1. The base pair sub­stitution at the third base pair of OR 1 was found to greatly reduce the affinity of OR I for A.-Cro (6). Furthermore, another substitution study showed that the A.-Cro prefers OR3 which is T at position 3, and A.-Cro distinguishes the OR3 from OR 1 primarily on the basis of the difference at position 3 ( 1 0). In their model, Asn31 has a contact with Thy3 (10). In our model of the A.-Cro-SYMG 17-mer complex, the methyl group of thymine at the third base pair is situated near the sidechain methylene groups of Asn31. The contact of Asn31 with Thy 3 has been also shown in the crystal structure (17, 18). Since the low sensitivity of the 13C filtered exper­iments prevented this inter-molecular NOE from being detected, it is not clear that this contact is the real in the solution structure.

The lowest affinity of A.-Cro for OR2 among three OR operators can be attributed to the additional base substitution at the first base pair. The methyl group of the thymine at the first base pair of the consensus sequence forms a hydrophobic con­tact with the methyl group of Thr17 as indicated by intermolecular NOEs. Removal of this hydrophobic contact is expected to reduce the affinity for the OR2. Consequently, the reduction of hydrophobic contacts described above explains the order of affinity of operator DNA to A.-Cro.

Conformation of the DNA and A.-Cro in the Complex

The crystal structure of the A.-Cro-DNAcomplex indicated that the conformation of the bases involved in protein recognition (residues 1-6 and 12-17) are typical B­form and that the DNA is bent at an angle of about 40 degrees (17,18,44). This bending of DNA is consistent with our NMR studies that the chemical shift of imino protons revealed bending or distortion at the center of the DNA in the A.-Cro­OR3 complex (13). Furthermore, the pattern of intra-molecular NOEs of the DNA in the A.-Cro-SYMG 17-mer complex was typical for that of B-form DNA at residues 1-6 and 12-17. However, extensive line broadening, probably due to con­formational exchange around the central mismatch base pair, caused many of the NMR signals from residues 7-11 to disappear. Therefore, few distant constraints were obtained for these residues and their precise conformation was undetermined by only use of the NMR data.

The bending of the OR3 17-mer upon complex with A.-Cro has been suggested by chemical modification experiments (11). GC9, which is the central base pair of the OR3 17-mer, is known from their results to be reagent-accessible in the complex with A.-Cro, namely GC9 is not covered by A.-Cro. While the DNA was bent at an

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Contacts Between the A.-Cro Repressor

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Figure 4: Stereo view of a best-fit superposition of calculated 20 structures of the complex of 1..-Cro protein and SYMG 17-mer.lndividual peptide backbones (N, Ca, C') of the 1..-Cro dimer are shown in blue or yellow. Nucleic acids are shown in white. This figure was made using MIDAS Plus program (Ferrin, et al., 1988).

Figure 5: Ribbon diagram of a representative structure of calculated structures of the 1..-Cro-SYMG complex. The peptide backbone is shown by blue or yellow ribbon, and the nucleic acid is indicated by white stick. This figure was made using QUANTA software (Molecular Simulations Inc.).

Figure 6: Representation of the hydrophobic interaction observed between 1..-Cro and SYMG 17-mer in complex. Thr17, Val25, and Ala29 are shown as yellow stick on rib­bon diagrams of the peptide backbone. The methyl groups of these residues are shown by van der Waals representations. Thymine! and Thymine13 in the DNA are colored by white and other nucleic acid residues are colored by gray. The methyl groups of Thymine! and Thyminel3 are also shown by van der Waals representations. This fig­ure was made using QUANTA software (Molecular Simulations Inc.).

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angle of 40 degrees in the crystal structures ( 17,18,44 ), similar bent angle was also observed by gel electrophoresis using A.-Cro bound 21-base pair oligonucleotides containing the OR3 sequence ( 46). Scanning force microscopy showed that the bent angle induced by binding of A.-Cro to the specific site was 69° ± 11 o ( 4 7). They also pointed out that using very short oligonucleotides likely to yield a minimum esti­mation of the bent angle as they used 1 K base pairs, while other methods used 17-21 base pairs (47). Therefore, we adopted a bent complex model in solution for A.­Cro and the B-form DNA as found in the crystal structure (44). The calculated model satisfied all of the intra-protein, intra-DNA and inter-molecular distance constraints with no distant violation greater than 0.5A despite the overall shape of the DNA being constrained to the bent B-form of the crystal structure. This model indicates a possible form of the A.-Cro-DNA complex in solution.

The structure of the A.-Cro monomer had been well defined by using NMR data. Despite extensive chemical shift changes, the structure of the A.-Cro monomer did not change drastically upon binding. The three a helices and one ~ sheet com­prised of three anti-parallel ~ strands were preserved upon binding as with the spa­tial arrangement of the secondary structural elements of residues 3 to 59. The C­terminal region (residue 60-66) was disordered in unbound A.-Cro ( 19). The crystal structure of the complex revealed that the Ser60 to have hydrogen bonds to a phos­phate group, Asn6l to be positioned within the minor groove, and reminder of the tail to be disordered (17,18,51). Using NMR, we confirmed that the C-terminal region of A.-Cro contacts the DNA upon binding. When an asymmetric operator DNA (containing 21 base pairs with the OR3 sequence) was complexed with A.-Cro, Thr64 gave two IH_I5N HSQC cross peaks (Figure 7). Each cross peak came from a different A.-Cro monomer subunit since their interaction with the asymmetric DNA caused them to be in different environments. Furthermore, in the absence of SYMG 17-mer, the IH_I5N HSQC cross peaks ofThr64 and Thr65 were absent due to fast exchange at 45°C and pH 7. However, these cross peaks appeared upon binding to the SYMG 17-mer. Clearly, the amide exchange was suppressed upon binding to the DNA. Furthermore, the chemical shift of the amide proton of Ser60 was shifted dramatically downfield upon complex formation. In the model, this amide proton is close to the phosphate group ofThyl3 of the SYMG 17-mer and can easily form a hydrogen bond. The location of the amide proton of Ser60 is sup­ported by an inter-subunit NOE with the y-methyl group of Val25 that has several inter-molecular NOEs to Thy 13. These observations clearly indicate that the C-ter­minal region of the A.-Cro interacts with the DNA in the complexed form.

r,t%~ ·<',, . ; .n ~7

- 114

115

Thr65 116

117

118

9.0 8.8 8.6 8.4 8.2 8.0 Hppm

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Contacts Between the A.-Cro Repressor

and the Operator DNA

Figure 7: A region of 2D IH-IsN HSQC spectrum of the uniformly ISN-Iabeled A.-Cro protein with the OR3 21-mer at 35°C. The ratio of DNAIA-Cro dimer is 1.2. Sample contains 0.5 mM protein in 50 mM KCI and 20 mM phosphate buffer (H20/2HP ratio 9: I, pH 6.8). The assignments are indicated by the amino acid numbers.

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Stabilization of the A.-Cro Protein Upon DNA Binding

The amide IHJ2H exchange rates of A.-Cro were much slower in the bound form than in the DNA free form (Figure 3). In order to exchange to deuterons, the back­bone amide protons within regions of regular secondary structural elements must break their hydrogen bonds and hence amide exchange rates are a measure of pro­tein rigidity. The increased rigidity within the secondary structural elements in the complexed form arises because the two subunits of A.-Cro become fixed upon bind­ing. The fluctuation of the free protein between the dimeric and monomeric states desists upon DNA binding causing the tertiary structure of A.-Cro to be more stable in the complexed form. This suppression of dimer-dissociation is probably the cause of the thermal stabilization of A.-Cro upon complexation. While the DNA free A.-Cro is unstable at temperatures higher than 30°C, the A.-Cro-operator complex was very stable up to 50°C. The thermodynamics of the A.-Cro-DNA interactions have been studied using pulsed-flow microcalorimetry (7) and the thermal denatu­ration profile of both DNA free A.-Cro and the A.-Cro-DNA (17-mer with OR3 sequence) complex was monitored by DSC (differential scanning calorimetry). While the melting of free A.-Cro starts at 25°C, the A.-Cro-OR3 complex remains sta­ble up to 70°C. The increased rigidity of the A.-Cro protein upon DNA binding is consistent with the increased thermal stability upon binding.

Increased rigidity and thermal stability were also observed for the V55C mutant version of A.-Cro relative to the native protein. In the V55C mutant protein, the valine at position 55 was substituted for a cysteine introducing a disulfide bond between the two subunits of A.-Cro. This mutant had much slower amide exchange rates than the wild type A.-Cro (Sato, S., personal communication). Furthermore, while the melting temperature, Tm, of free wild type A.-Cro was 46.4°C, the V55C mutant had a Tm of 54.5°C (52).

In summary, we have characterized the inter-molecular contacts between the A.-Cro and the operator DNA using NMR. We employed a symmetric consensus sequence to make the system simple and feasible for NMR analysis. However, this modification caused the disappearance of signals from protons located in close vicinity to a mismatched central base pair. Hence, the structural characterization of the DNA, by NMR alone, proved to be a great challenge. Numerous studies reveled that DNA bends upon binding A.-Cro in solution. Hence, we adopted a bent B-form of DNA as found in the crystal structure to generate a possible model using the simulated annealing method. This hybrid model con­firmed interactions implied by inter-molecular NOEs and revealed the role of each of these interactions in the operator recognition of A.-Cro. Furthermore, the model suggest­ed other interactions whose inter-molecular NOEs might not be observed due to the low sensitivity of the isotope filter experiments. The rather extended structure and reduced number of protons of DNA compared to proteins causes it to be a considerable challenge to NMR spectroscopists. Recently, it has become possible to produce isotope labeled DNA and most likely this will become more popular in the future. However, in cases where the general structure of the DNA has been determined by other methods, our approach is both powerful and time saving. Furthermore, this approach could be extend­ed to study protein/protein interactions as many protein structures are available from data bank. However, it is crucial, when using this approach, that the NOE data confirms the model. It is important that the model does not bias the analysis of the NOE data. We used our NOE data to ensure our model is correct and to position the DNA onto the protein.

Acknowledgements

The authors thank Dr. Koichi Uegaki for the helpful discussions. This work was supported by a Grant-in-Aid for Basic Scientific Research, Category B (No.09580176), from the Ministry of Education, Science and Culture, Japan, and the Human Frontier Science Program (RG-401195).

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Date Received: December 31, 1998

Communicated by the Editor Ramaswamy H. Sarma

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