Paper Jose Delgado

Published online 11 December 2008 Nucleic Acids Research, 2009, Vol. 37, No. 2 629637doi:10.1093/nar/gkn976

The structures of non-CG-repeat Z-DNAsco-crystallized with the Z-DNA-binding domain,hZaADAR1Sung Chul Ha1, Jongkeun Choi1, Hye-Yeon Hwang1, Alexander Rich2,

Yang-Gyun Kim3,* and Kyeong Kyu Kim1,*

1Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan UniversitySchool of Medicine, Suwon 440-746, Korea, 2Department of Biology, Massachusetts Institute of technology,Cambridge, Massachusetts 02139, USA and 3Department of Chemistry, Sungkyunkwan University, Suwon440-746, Korea

Received October 17, 2008; Revised and Accepted November 19, 2008

ABSTRACT

The Z-DNA conformation preferentially occurs atalternating purine-pyrimidine repeats, and is specifi-cally recognized by Za domains identified in severalZ-DNA-binding proteins. The binding of Za toforeign or chromosomal DNA in various sequencecontexts is known to influence various biologicalfunctions, including the DNA-mediated innateimmune response and transcriptional modulationof gene expression. For these reasons, understand-ing its binding mode and the conformational diver-sity of Za bound Z-DNAs is of considerableimportance. However, structural studies of Zabound Z-DNA have been mostly limited to standardCG-repeat DNAs. Here, we have solved the crystalstructures of three representative non-CG repeatDNAs, d(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2complexed to hZaADAR1 and compared those struc-tures with that of hZaADAR1/d(CGCGCG)2 and theZa-free Z-DNAs. hZaADAR1 bound to each of thethree Z-DNAs showed a well conserved bindingmode with very limited structural deviation irrespec-tive of the DNA sequence, although varying numbersof residues were in contact with Z-DNA. Z-DNAsdisplay less structural alterations in the Za-boundstate than in their free form, thereby suggestingthat conformational diversities of Z-DNAs arerestrained by the binding pocket of Za. These datasuggest that Z-DNAs are recognized by Za through

common conformational features regardless of thesequence and structural alterations.

INTRODUCTION

DNA can adopt various secondary conformations otherthan the classical right-handed B-DNA structure undercertain specic physiological conditions (1). Left-handedZ-DNA has been studied in detail by numerous methodsover the last two decades since its rst crystal structurewas solved (2). Z-DNA conformations in many dier-ent sequences have been a challenge to crystallize.Nonetheless, several structures have been determined byX-ray crystallographic study (3). Many characteristic fea-tures of the Z-DNA structure have been identied fromthe accumulated structural data. The overall shapes ofmost Z-DNA structures share common features that aresimilar to those seen in the rst crystallized Z-DNA struc-ture of d(CGCGCG)2 (2). Z-DNA has a zig-zag sugarphosphate backbone and is longer and thinner thanB-DNA. Its nucleotides form in a dinucleotide repeat inwhich they alternate with syn and anti conformations.Z-DNA is favored in alternating pyrimidine-purine(APP) sequences (4). The alternating CG-repeat sequenceis the most favorable energetically for Z-DNA formation(5). Networks of water molecules hydrate Z-DNA, form-ing hydrogen bonds with atoms in both the backbone andbase. However, some Z-DNAs form with dierent struc-tural features, especially those with sequences withoutAPP or including A-T base pairs (68). When A-T basepairs are introduced, the overall Z-DNA structure

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes of hZaADAR1 in complex withd(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2 are 3F21,3F22 and 3F23, respectively)

*To whom correspondence should be addressed. Tel: +82 31 299 6136; Fax: +82 31 299 6159; Email: [email protected] may also be addressed to Yang-Gyun Kim, Tel: +82 31 299 4563; Fax: +82 31 299 4575; Email: [email protected]

2008 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

by guest on April 26, 2015

http://nar.oxfordjournals.org/D

ownloaded from

becomes partially distorted due to disruption of the hydra-tion spine (6). In addition, the non-APP base pairs havebeen found to be highly buckled when compared withother base pairs in Z-DNA (7,8). Up to now, however,there have been only a limited number of structural studiesof Z-DNAs containing non-APP or A-T base pairs. Moststudies have been carried out in the presence of excesscations or using dsDNAs modied by methylation or bro-mation. Thus, study of Z-DNA under low salt conditionsor in the presence of Z-DNA-binding domains (Za)remains to be explored, and this may provide insightinto the eect that sequence variability has on Z-DNAconformation under physiological conditions.The protein Za domain was rst identied from human

ADAR1 (double-stranded RNA adenosine deaminase)and subsequently found in other proteins (DLM1, E3Land PKZ). These domains provide a unique opportunityto explore Z-DNA and its various roles in biological sys-tems (913). The Za domains are highly specic for the Z-conformation of nucleic acids, including dsDNA, dsRNAas well as DNARNA hybrids. They have bind-ing anities in the low-nanomolar range. In the crystalstructures of double-stranded d(CGCGCG)2 complexedwith hZaADAR1, mZaDLM1, yabZaE3L or hZbDLM1(10,11,1416), a close resemblance was found betweenthe bound and unbound states of the Z-DNA structureof d(CGCGCG)2 (2). It is now known that Z-DNA isfound in the genome as an active transcription modierthat functions by modulating chromatin structure (17,18).The Z-DNA conformation is not limited only to APPsequences, but can appear in many other nucleotidesequences. Recent functional studies of the Z-DNA-bind-ing domains in vaccinia E3L showed that it acts as a tran-scription modulator of several apoptosis-related genes(19). More recently, the Z-DNA-binding protein DLM1has been found to act in the innate immune system as acytosolic receptor for dsDNA, recognizing foreign patho-genic DNA (20). These results support the idea thatdiverse sequences of Z-DNA can be recognized byZ-DNA-binding domains and that their binding is essen-tial for cellular processes. To understand the bindingmode of Za to Z-DNAs in various sequence contexts, itis necessary to investigate structural features of Z-DNAsbound to Za and to compare their structures withZ-DNAs stabilized by base-modication and/or positivelycharged ions. In this regard, we undertook a study to solveZ-DNA structures with non-CG-repeat sequences stabi-lized by the same Za domain. Here we report the co-crys-tal structures of three non-CG-repeat Z-DNAs containingeither A-T base pairs or non-APP sequence bound tohZaADAR1. These complexes reveal how the structuraldiversity of Z-DNA caused by non-CG-repeat sequencesis recognized and stabilized by the Z-DNA-bindingdomain.

MATERIALS AND METHODS

Expression and purification

hZaADAR1 (residues 133209) from human ADAR1 wasexpressed and puried as described previously (21).

In brief, hZaADAR1 was puried through sequential chro-matographic steps involving HiTrap metal anity column(GE Healthcare, Piscataway, NJ), thrombin digestion forthe removal of N-terminal his-tag and a ResourceS column (GE Healthcare, Piscataway, NJ). The purityand concentration of hZaADAR1 were estimated by SDSPAGE and the Bradford method, respectively. DNAswere purchased (IDT, Coralville, IA) and puried asdescribed previously (22).

Crystallization

The DNA used for crystallization all had 6nt, plus a 50 Toverhang which acts as a stabilizing capping residue. Forcrystallization, hZaADAR1 was mixed with dsDNA [(d(TCGCCCG)::d(TCGGGCG), d(TCACGTG)2, d(TCGTACG)2 or d(TCGGCCG)2)] at a 0.66mM : 0.33mM molarratio in 5 mM HEPES-NaOH pH 7.5 containing 20mMNaCl, and incubated at 303K for at least 2 h. All crystal-lization experiments were performed using the hangingdrop vapor diusion method with a VDX plate at295K. Among the four dsDNA used in crystallization,only d(TCGCCCG)::d(TCGGGCG) was successfullyco-crystallized with hZaADAR1 when ammonium sulfatewas used as precipitant. For crystallization of the otherthree complexes, crystals of hZaADAR1/d(TCGCCCG)::d(TCGGGCG) were used as seeds, and initial crystalswere again used as seeds for further crystallization.Diraction quality crystals were obtained within amonth using 2.2M ammonium sulfate and 10% glycerolin the reservoir solution.

Data collection and structure determination

Preliminary X-ray diraction analyses were performed atbeamline BL6A of PAL (Pohang, Korea). X-ray dirac-tion data of frozen crystals were collected at 100K with aMAR CCD165 detector at the BL41-XU beamline ofSpring-8 (Harima, Japan). Crystals were frozen either inliquid nitrogen directly or by using paraton as a cryopro-tectant. Diraction data were processed and integratedusing HKL2000 (23). The unit cell parameters, spacegroup and other data collection statistics are summarizedin Supplementary Table 1. The crystal structure ofhZaADAR1/d(TCGCGCG)2 (PDB ID 1QBJ) was usedfor the initial model of the other complex structures.Renement and model building were performed by CNS(24) and O (25), respectively. The renement statistics aresummarized in Supplementary Table 1. The crystal struc-ture of hZaADAR1/d(TCGCCCG)::d(TCGGGCG) wasnot rened because of ambiguity in base assignment.All gures were drawn using Molscript, Raster3D andPymol (26,27, http://www.pymol.org). The structuralsuperposition was performed using the LSQKAB CCP4program (28).

RESULTS AND DISCUSSION

Structure determination

The crystal structures of hZaADAR1 complexed with threenon-CG-repeat dsDNAs, d(TCACGTG)2, d(TCGTACG)2

630 Nucleic Acids Research, 2009, Vol. 37, No. 2



ownloaded from

and d(TCGGCCG)2 were determined at resolutions of2.2, 2.5 and 2.7 A, respectively (SupplementaryTable 1). The simulated annealed omit maps contouredat 3s near the altered sequences conrmed that currentstructural information represents each non-CG-repeatDNA (Supplementary Figure 1). In all three structures,the deoxythymidine overhangs at the 50 end of the oligo-nucleotides were not modeled due to their weak electrondensities, and therefore, are not mentioned in currentstudy. There are three hZaADAR1 domains, assigned aschains A, B and C, and three DNA strands, chains D, Eand F, in one asymmetric unit. Chains D and E form theZ-DNA duplex, and chain F forms a duplex with chainF in another asymmetric unit that is related by crystal-lographic 2-fold symmetry. Each DNA strand is boundto one hZaADAR1 domain. The protein/DNA complexmade up of the chain C and chain F pair was used forstructural analyses unless specied otherwise because itsaverage temperature factor is the lowest among the threecomplexes in the same asymmetric unit.

Overall structures

Overall, the hZaADAR1 domains bound to the three dier-ent Z-DNAs used in this study have almost identical struc-tures to that of hZaADAR1 bound to d(CGCGCG)2 (14).hZaADAR1 has an a/b topology containing a three-helixbundle anked on one side by a twisted antiparallel bsheet. The root mean square deviations (RMSDs) betweenhZaADAR1 bound to d(CGCGCG)2 and hZaADAR1 boundto d(CACGTG)2, d(CGTACG)2 or d(CGGCCG)2 are0.52, 0.19 and 0.19 A, respectively, when calculated usingthe 64 Ca atoms of hZaADAR1 (Figure 1A). The threeZ-DNAs bound to hZaADAR1 are all in theZ-conformation with alternating anti-and syn-glycosidicbonds regardless of their sequence (Figure 1B). With theexception of purines G3 and pyrimidines C4 of d(CGGCCG)2 that adopt the anti and syn conformations, respec-tively, all other purines and pyrimidines are in the synand anti conformations, respectively (Figure 1B). The cal-culated double-stranded DNA parameters are within therange of a Z conformation of DNA (SupplementaryTable 2). For Z-DNAs complexed with hZaADAR1, theRMSD values between d(CGCGCG)2 and other DNAs,d(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2 were0.28, 0.33 and 0.56 A, respectively, when calculated with63 DNA backbone atoms of chain F for each DNA struc-ture (Figure 1B and Supplementary Table 3).

Interactions between hZaADAR1 andnon-CG-repeat Z-DNAs

The structural analyses of hZaADAR1, mZaDLM1 andyabZaE3L bound to the Z conformation of d(CGCGCG)2revealed well-conserved interactions with the Za domains(10,11,14). Za domains with a winged helixturnhelix motif bind to Z-DNA in a conformation-specic manner. The residues in the recognition helix (a3)and in the wing play critical roles in binding Z-DNAthrough direct or water-mediated hydrogen bonds andvan der Waals interactions (Figure 2). On Z-DNA,

one continuous surface composed of the sugar-phosphatebackbone of Z-DNA is mostly involved in protein contact.Similarly, in the crystal structures of hZaADAR1 bound

to the three non-CG-repeat dsDNAs the residues locatedin a3 and the wing mostly make contact with the Z-DNAbackbones either directly or through water-mediatedinteractions (Figure 2). When the DNA-binding surfacesof the hZaADAR1 domains bound to the four Z-DNAs arecompared, their curvatures and surface charge distribu-tions are nearly identical (Figure 3). These results,together with the limited structural alterations found inhZaADAR1 domains, strongly demonstrate that the overallbinding mode of hZaADAR1 to Z-DNA are well conservedregardless of the sequence of the bound Z-DNA.The number and type of hZaADAR1 residues contacting

bases on each DNA vary, mainly because some of theimportant residues that were identied as the DNA-contacting residues in hZaADAR1/d(CGCGCG)2 (14) aredisordered (Figure 2). However, it does not appears thatvariations in protein/DNA contacts are related to thesequence of the Z-DNA, since the number of contactingresidues is not the same, even among the three hZaADAR1domains in one asymmetric unit of each ZaADAR1/DNAcrystal. For example, in most structures, the roles ofArg174 and Thr191 are neither well dened nor involvedin DNA contact. In hZaADAR1/d(CGCGCG)2, Arg174was shown to form a direct hydrogen bond to a phosphateatom and a water-mediated hydrogen bond to the ribosering (14). However, in seven of the nine hZaADAR1domains used in this study, the amine groups of Arg174are not modeled due to their weak electron densities(Figure 2). In the case of Thr191, no DNA interactionhas been found. Similarly, Lys169 and Lys170 are onlyin contact with DNA in some structures (Figure 2). Incontrast, Asn173, Tyr177, Pro192, Pro193 and Trp195of hZaADAR1 contribute to the recognition of Z-DNA inall cases. Asn173 and Tyr177, located in the recognitionhelix (a3), recognize the phosphate backbone of Z-DNAthrough direct or water-mediated hydrogen bonds asobserved in hZaADAR1/d(CGCGCG)2 (14). Likewise,Pro192 and Pro193 in the wing interact with Z-DNAthrough van der Waals interactions. In some cases,Trp195 makes a water-mediated hydrogen bond to a phos-phate, but its major role seems to be supporting Tyr177via the hydrophobic edge-to-face interaction which stabi-lizes the interactions between Tyr177 and the bases in theN4 position (Figure 2). Moreover, some coordinatedwaters that are well dened and mediate hydrogenbonds between protein and DNA in the crystal structureof hZaADAR1/d(CGCGCG)2 are not found in somestructures.The decrease in protein/DNA interactions in the three

complex structures compared to hZaADAR1/d(CGCGCG)2 can be explained in part by the decrease in dirac-tion resolution. However, it seems that proteinDNAinteractions are not all aected by diraction resolutionsince well-dened interactions are consistently observed inall three structures despite their resolution dierences. Forexample, the electron density of the well-dened hydrogenbonds between Tyr177 and the phosphate group of G3 arevery clear even in the case of hZaADAR1/d(CGGCCG)2

Nucleic Acids Research, 2009, Vol. 37, No. 2 631



ownloaded from

whose structure was determined at 2.7 A resolution(Supplementary Figure 2). Conversely, it is suspectedthat some of the unseen protein/DNA interactions fromthese three Z-DNA structures are neither strong nor essen-tial for Z-DNA recognition. From these results, it can behypothesized that some of the residues previously identi-ed as Z-DNA binders are not indispensable for Z-DNAbinding but that they have auxiliary roles in DNA bindingand probably produce tighter binding. However, wecannot rule out the possibility that crystal packing forcesmay have aected or destabilized some of the interactionsbetween hZaADAR1 and Z-DNA.

In the Za/d(CGCGCG)2 structure as well as in the cur-rent crystal structure of hZaADAR1/d(CATGCG)2, the tyr-osine residue of the recognition helix (a3) has a uniquerole in recognizing the C-8 carbon of the syn deoxygua-nosine at the forth position (G4) via a CHp interaction(10,11,14; Figure 2A). In the case of Za/d(CGTACG)2,the deoxyadenosine (A4) also adopts the syn conforma-tion, which was very similar to that observed in previousstudies (Figure 2B). More interesting is the observationthat hZaADAR1 stabilizes the syn conformation of deoxy-cytidine at the forth position (C4) of d(CGGCCG)2(Figure 2C). Generally, the syn conformation is not

Figure 1. Structural comparison of the hZaADAR1 domain and the Z-DNAs in several complexes. (A) Overlapping Ca traces of the hZaADAR1domains when complexed to d(CGCGCG)2 [cyan], (NDB ID PH0001), d(CACGTG) 2 [red], d(CGTACG)2 [magenta] and d(CGGCCG)2 [green].Sixty-four Ca atoms of chain C in each structure were used for the superposition. The N- and C-termini and a3 are labeled. (B) Four Z-DNAstrands, CACGTG (red), CGTACG (magenta), CGGCCG (green) and CGCGCG (cyan), bound to hZaADAR1 were compared by superimposingfour Za/Z-DNA complexes using 64 Ca atoms of hZaADAR1 domains. In order to show the relative orientation of the Z-DNA and Za, chain C ofthe hZaADAR1/d(CGCGCG)2 complex was drawn in a ribbon diagram. The amino acid residues and water molecules involved in DNA binding aredrawn as sky blue stick models and green balls, respectively. Arg174 and Thr191 are marked by yellow stick models to dierentiate them from othercore resides. These residues are mostly disordered in the current study, but are well dened and bind to DNA in the crystal structure of thehZaADAR1/d(CGCGCG)2 complex.




ownloaded from

Figure 2. The proteinDNA interactions in three dierent Za-non-alternating CG ZDNA complexes. (A) hZaADAR1/d(CACGTG)2, (B) hZaADAR1/d(CGTACG)2 and (C) hZaADAR1/d(CGGCCG)2. One representative Za/DNA complex (chains C and F) of the three complexes in one asymmetricunit is drawn as a tubular ribbon diagram (left). The three complexes in the asymmetric unit are then shown as schematic diagrams (left to right).These are chains A and D, chains B and E, and chains C and F, respectively. In the schematic diagrams, the amino acids identied as the DNA-contacting residues in the structure of the hZaADAR1/d(CGCGCG)2 complex are marked by boxes. Disordered residues are indicated by dottedboxes. Hydrogen bonds are represented by dotted lines and van der Waals contacts by open circles. The CHp interaction between the conserved Tyrresidue and syn deoxynucleoside is indicated by lled circles. Waters are shown as gray circles. In the tubular ribbon diagrams, the same residuesused in the schematic diagrams are drawn as stick models and labeled. The DNA backbones and labeled bases are shown as red and gray stickmodels, respectively. Water molecules are shown as green spheres. Hydrogen bonds are drawn as dashed lines.




ownloaded from

favored for pyrimidine nucleotides unless they aremodied (3,4). However in the crystal structure ofhZaADAR1/d(CGGCCG)2, the syn conformation of deox-ycytidine is stabilized by the CHp interaction between theC-5 and C-6 carbons of C4 and the p orbital of the tyr-osine ring (Figure 2C). These data show that the bindingmode of hZaADAR1 observed for d(CGCGCG)2 is also atemplate for non-CG-repeat Z-DNAs containing non-APP repeat sequences or A-T base pairs. As with the ear-lier structure, there are no sequence-specic interactions

between Za and Z-DNA. These results reinforce the ideathat the binding of Za to Z-DNA is sequence-independentbut conformation specic.

Structural variations between free andhZaADAR1-bound Z-DNAs

The crystal structures of d(CGCGCG)2, d(CACGTG)2,d(m5CGTAm5CG)2 and d(

m5CGGCm5CG)2 were used torepresent Z-DNAs free of protein binding (SupplementaryTable 3; 2,8,29,30), and they have been compared with thehZaADAR1-bound Z-DNA structure. In order to stabilizeand crystallize Z-DNA with non-APP or A-T base pairs,base modication is used or it is necessary to add cationssuch as metal ions or polyamines. The 5 position of cyto-sine was methylated for the crystallization of Z-form d(CGTACG)2 and d(CGGCCG)2 (8,29), and spermine wasadded for the crystallization of Z-form d(CGCGCG)2and d(CACGTA)2 (2,30). When the protein-free andhZaADAR1-bound Z-DNAs were compared by superim-posing each DNA pair using 63 DNA backbone atoms,RMSDs of d(CGCGCG)2, d(CACGTG)2, d(CGTACG)2and d(CGGCCG)2 pairs were 0.89, 0.75, 0.57 and 0.61 A,respectively (Figure 4 and Supplementary Table 3).Regardless of their sequence, the main structural dier-ences between the free and bound Z-DNAs were foundin the helical rise: when bound to hZaADAR1 it increasedin the N3pN4 step, but decreased in the N2pN3 andN4pN5 steps (Supplementary Table 2). However, the dis-tance changes in the N1pN2 and N5pN6 steps occurredregardless of Za binding (Supplementary Table 2).Structural variations between protein-free and

hZaADAR1-bound Z-DNAs were compared directly byplotting the distance between two corresponding atomsin Z-DNAs along the DNA backbone atoms(Figure 5A). In this manner, the structures of non-CG-repeat Z-DNAs and CG-repeat Z-DNA were also ana-lyzed (Figure 5BD). Structural deviations are plottedfor all comparisons of protein-free and Za-boundZ-DNA sequences. The largest structural variationbetween protein-free and hZaADAR1-bound Z-DNAs wasdetected in the two phosphates at the N2pN3 and N4pN5phosphodiester steps, respectively. These results wereexpected because the ZI conformation of the GpC

Figure 4. Structural comparisons of free- and hZaADAR1-bound Z-DNAs. Superposition of the free d(CACGTG)2 (blue) with Za-boundd(CACGTG)2 (red) (A), the free d(

m5CGTAm5CG)2 (cyan) with Za-bound d(CGTACG)2 (magenta) (B) and the free d(m5CGGCm5CG)2 (yellow)

with Za-bound d(CGGCCG)2 (green) (C). For the structural overlap, 63 DNA backbone atoms of chain F of each ZaADAR1-bound Z-DNA andchain A of each free Z-DNA were used. The Ca trace of hZaADAR1 in each complex is drawn in gray. The N- and C- termini, recognition helix (a3)of hZaADAR1 and the 50 and 30 ends of the DNA are labeled.

Figure 3. Surface charge distributions of the hZaADAR1 domains com-plexed with various Z-DNAs, as viewed along the DNA bindingcleft. DNA-binding surfaces of the hZaADAR1 domains bound tod(CGCGCG)2 (upper left), d(CACGTG)2 (upper right), d(CGTACG)2 (bottom left) and d(CGGCCG)2 (bottom right) are drawn,and their surface charge distributions are displayed. Arg174 andThr174 were not used for the surface charge calculations since theyare not well dened in most structures. The red and blue areas repre-sent the negatively and positively charged surfaces, respectively. DNAbackbones are shown in stick models with phosphate atoms in yellow,oxygen in red, nitrogen in blue and carbon in gray.




ownloaded from

phosphodiester step is preferred when Z-DNA forms acomplex with hZaADAR1 due to the specic interactionbetween the phosphate groups of DNA and the chargedresidues of Za, whereas protein-free Z-DNA can have twoalternative conformations, ZI and ZII (14,31). The dier-ence between two corresponding atoms of the comparedZ-DNAs is >2.5 A when the protein-free Z-DNA is in theZII conformation, and it is

Z-DNAs containing non-APP sequences or A-T basepair(s) have intrinsic structural instability and anincreased solvent-exposed surface since base pairs arebuckled out of the base-pair plane and protrude into themajor groove (3,8,30,31). These structural featuresare still observed in hZaADAR1-bound Z-DNAs(Supplementary Table 2). For example, d(CGTACG)2and d(CGGCCG)2 reveal a huge buckle and decreasedstacking interactions, whether bound to Za or not(Figure 4 and Supplementary Table 2). Therefore, it isthought that the base packing pattern and structural insta-bility of non-CG-repeat Z-DNAs are always maintained,whereas the phosphate backbone conformations arerestrained by the Z-DNA-binding pocket of hZaADAR1.Overall, additional structural instability of the Z confor-mation caused by non-CG-repeat sequences did not sig-nicantly aect the maintenance of the Z conrmation ofDNAs in their complexes with Za. These results suggestthat more energy is gained upon Za binding than isneeded to overcome the structural instability of non-CG-repeat Z-DNAs.

CONCLUSIONS

Our structural data strongly support the idea thathZaADAR1 recognizes Z-DNA in a conformation-specicmanner, but not in a sequence-specic manner. The struc-tures of hZaADAR1 complexed with three dierent non-CG-repeat double-stranded Z-DNAs (with non-APPsequences or A-T base pairs) reveal that hZaADAR1 bindsand stabilizes the Z conformation of DNA via a similarbinding mode to that of the Za/d(CGCGCG)2 complex,regardless of sequence context. Most notably, the CHpinteraction was observed between Tyr177 and the cytosinein the syn conformation. While structures of phosphatebackbones in free and hZaADAR1-bound states displaysome variation near each phosphate group, hZaADAR1does not exert a profound eect on the Z-DNA base pairparameters induced by incorporation of non-APP and A-Tbase pairs. Irrespective of the heterogeneity in sequenceand structure of non-CG-repeat Z-DNAs, Za recognizesthe DNA backbone atoms and imposes structural restraintthrough the core residues located on the DNA-bindingsurface. It has been suggested that dierent Z-DNA-bind-ing proteins can bind to chromosomal or foreign DNAsand take part in essential biological processes. In this con-text, the Za domain is expected to recognize and stabilizestretches of DNA in the Z-DNA conformation. Our resultsprovide a molecular basis for understanding how the Zaproteins recognize and stabilize Z-DNAs in varioussequence contexts.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

The Korea Science and Engineering Foundation(KOSEF) through the National Research Laboratory

Program funded by the Ministry of Education, Scienceand Technology (NRL-2006-02287). Funding for openaccess charges: the National Laboratory Program grantof the Korean government (MEST).

Conict of interest statement. None declared.

REFERENCES

1. Rich,A. (1993) DNA comes in many forms. Gene, 135, 99109.2. Wang,A.H., Quigley,G.J., Kolpak,F.J., Crawford,J.L.,van Boom,J.H., van der Marel,G. and Rich,A. (1979) Molecularstructure of a left-handed double helical DNA fragment at atomicresolution. Nature, 282, 680686.

3. Ho,P.S. and Mooers,B.H.M. (1997) Z-DNA crystallography.Biopolymers, 44, 6590.

4. Rich,A., Nordheim,A. and Wang,A.H. (1984) The chemistry andbiology of left-handed Z-DNA. Annu. Rev. Biochem., 53, 791846.

5. Ho,P.S. (1994) The non-B-DNA structure of d(CA/TG)n does notdier from that of Z-DNA. Proc. Natl Acad. Sci. USA, 91, 95499553.

6. Wang,A.H., Hakoshima,T., van der Marel,G., van Boom,J.H. andRich,A. (1984) AT base pairs are less stable than GC base pairs inZ-DNA: the crystal structure of d(m5CGTAm5CG). Cell, 37,321331.

7. Schroth,G.P., Kagawa,T.F. and Ho,P.S. (1993) Structure andthermodynamics of nonalternating C.G base pairs in Z-DNA: the1.3-A crystal structure of the asymmetric hexanucleotided(m5CGGGm5CG).d(m5CGCCm5CG). Biochemistry, 32,1338113392.

8. Eichman,B.F., Schroth,G.P., Basham,B.E. and Ho,P.S. (1999)The intrinsic structure and stability of out-of-alternation base pairsin Z-DNA. Nucleic Acids Res., 27, 543550.

9. Herbert,A., Alfken,J., Kim,Y.G., Mian,I.S., Nishikura,K. andRich,A. (1997) A Z-DNA binding domain present in the humanediting enzyme, double-stranded RNA adenosine deaminase. Proc.Natl Acad. Sci. USA, 94, 84218426.

10. Schwartz,T., Behlke,J., Lowenhaupt,K., Heinemann,U. and Rich,A.(2001) Structure of the DLM-1-Z-DNA complex reveals a con-served family of Z-DNA-binding proteins. Nat. Struct. Biol., 8,761765.

11. Ha,S.C., Lokanath,N.K., Quyen,D.V., Wu,C.A., Lowenhaupt,K.,Rich,A., Kim,Y.G. and Kim,K.K. (2004) A poxvirus protein forms acomplex with left-handed Z-DNA: crystal structure of a YatapoxvirusZalpha bound to DNA. Proc. Natl Acad. Sci. USA, 101, 1436714372.

12. Hu,C.Y., Zhang,Y.B., Huang,G.P., Zhang,Q.Y. and Gui,J.F. (2004)Molecular cloning and characterisation of a sh PKR-like genefrom cultured CAB cells induced by UV-inactivated virus. FishShellsh Immunol., 17, 353366.

13. Rothenburg,S., Deigendesch,N., Dittmar,K., Koch-Nolte,F.,Haag,F., Lowenhaupt,K and Rich,A. (2005) A PKR-like eukaryoticinitiation factor 2alpha kinase from zebrash contains Z-DNAbinding domains instead of dsRNA binding domains. Proc. NatlAcad. Sci. USA, 102, 16021607.

14. Schwartz,T., Rould,M.A., Lowenhaupt,K., Herbert,A. and Rich,A.(1999) Crystal structure of the Zalpha domain of the human editingenzyme ADAR1 bound to left-handed Z-DNA. Science, 284,18411845.

15. Ha,S.C., Lowenhaupt,K., Rich,A., Kim,Y.G. and Kim,K.K. (2005)Crystal structure of a junction between B-DNA and Z-DNA revealstwo extruded bases. Nature, 437, 11831186.

16. Ha,S.C., Kim,D., Hwang,H.Y., Rich,A., Kim,Y.G. and Kim,K.K.(2009) The crystal structure of the second Z-DNA binding domainof human DAI (ZBP1) in complex with Z-DNA reveals an unusualbinding mode to Z-DNA. Proc. Natl Acad. Sci. USA, doi:10.1073/pnas.0810463106.

17. Liu,H., Mulholland,N., Fu,H. and Zhao,K. (2006) Cooperativeactivity of BRG1 and Z-DNA formation in chromatin remodeling.Mol. Cell. Biol., 26, 25502559.

18. Wang,G. and Vasquez,K.M. (2007) Z-DNA, an active element inthe genome. Front. Biosci., 12, 44244438.

19. Kwon,J.A. and Rich,A. (2005) Biological function of the vacciniavirus Z-DNA-binding protein E3L: gene transactivation and




ownloaded from

antiapoptotic activity in HeLa cells. Proc Natl Acad. Sci. USA, 102,1275912764.

20. Takaoka,A., Wang,Z., Choi,M.K., Yanai,H., Negishi,H., Ban,T.,Lu,Y., Miyagishi,M., Kodama,T., Honda,K. et al. (2007) DAI(DLM-1/ZBP1) is a cytosolic DNA sensor and an activator ofinnate immune response. Nature, 448, 501505.

21. Schwartz,T., Lowenhaupt,K., Kim,Y.G., Li,L., Brown,B.A. II,Herbert,A. and Rich,A. (1999) Proteolytic dissection of Zab, theZ-DNA-binding domain of human ADAR1. J. Biol. Chem., 274,28992906.

22. Schwartz,T., Shafer,K., Lowenhaupt,K., Hanlon,E., Herbert,A. andRich,A. (1999) Crystallization and preliminary studies ofthe DNA-binding domain Za from ADAR1 complexed toleft-handed DNA. Acta Crystallogr. D Biol. Crystallogr., 55,13621364.

23. Otwinowski,Z. and Minor,W. (1997) Processing of X-raydiraction data collected in oscillation mode. Methods Enzymol.,276, 307326.

24. Brunger,A.T., Adams,P.D., Clore,G.M., DeLano,W.L., Gros,P.,Grosse-Kunstleve,R.W., Jiang,J.S., Kuszewski,J., Nilges,M.,Pannu,N.S. et al. (1998) Crystallography and NMR system: a newsoftware suite for macromolecular structure determination. ActaCrystallogr. D Biol. Crystallogr., 54, 905921.

25. Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991)Improved methods for building protein models in electron density

maps and the location of errors in these models. Acta Crystallogr.A, 47, 110119.

26. Merritt,E.A. (1994) A program for photorealistic moleculargraphics. Acta Crystallogr. D Biol. Crystallogr., 50, 869873.

27. Kraulis,P. (1991) MOLSCRIPT: a program to produce bothdetailed and schematic plots of protein structures. J. Appl.Crystallogr., 24, 946950.

28. Collaborative Computational Project, Number 4. (1994) The CCP4suite: Programs for protein crystallography. Acta Cryst., D50,760763.

29. Wang,A.H., Gessner,R.V., van der Marel,G.A., van Boom,J.H. andRich,A. (1985) Crystal structure of Z-DNA without an alternatingpurine-pyrimidine sequence. Proc. Natl Acad. Sci. USA, 82,36113615.

30. Narayana,N., Shamala,N., Ganesh,K.N. and Viswamitra,M.A.(2006) Interaction between the Z-type DNA duplex and1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 Aresolution. Biochemistry, 45, 12001211.

31. Wang,A.J., Quigley,G.J., Kolpak,F.J., van der Marel,G.,van Boom,J.H. and Rich,A. (1981) Left-handed double helical DNA:variations in the backbone conformation. Science, 211, 171176.

32. Sadasivan,C. and Gautham,N. (1995) Sequence-dependent micro-heterogeneity of Z-DNA: the crystal and molecular structures ofd(CACGCG):d(CGCGTG) and d(CGCACG):d(CGTGCG).J. Mol. Biol., 248, 918930.




ownloaded from

Paper Jose Delgado

Documents

Transcript of Paper Jose Delgado