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Proteolytic Dissection of Zab, the Z-DNA-binding Domain of Human ADAR1

1999; Elsevier BV; Volume: 274; Issue: 5 Linguagem: Inglês

10.1074/jbc.274.5.2899

1083-351X

Thomas Schwartz, Ky Lowenhaupt, Yang‐Gyun Kim, Liyun Li, Bernard A. Brown, Alan Herbert, Alexander Rich,

RNA and protein synthesis mechanisms

Zα is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zα is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Zα is conserved in ADAR1 from many species; in each case, there is a second similar motif,Zβ, separated from Zα by a more divergent linker. To investigate the structure-function relationship ofZα, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zα is part of a domain, Zab, of 229 amino acids (residues 133–361 in human ADAR1). This domain contains both Zα and Zβas well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133–209), containing only Zα, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zβ, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zαand Zβ act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein. Zα is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zα is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Zα is conserved in ADAR1 from many species; in each case, there is a second similar motif,Zβ, separated from Zα by a more divergent linker. To investigate the structure-function relationship ofZα, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zα is part of a domain, Zab, of 229 amino acids (residues 133–361 in human ADAR1). This domain contains both Zα and Zβas well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133–209), containing only Zα, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zβ, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zαand Zβ act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein. Many protein domains that recognize DNA in both sequence- and conformation-specific manners have been characterized (for a review, see Ref. 1Lilley D.M.J. DNA-Protein: Structural Interactions. Oxford University Press, Oxford1995Google Scholar). These studies have resulted in an understanding of the variety of ways in which protein-DNA interactions can result in function. Identification of a peptide motif, Zα, which binds specifically to Z-DNA, opens up a new vista and invites the investigation of the similarities and differences between domains that bind right- and left-handed DNAs. The conformation specificity ofZα binding has been characterized in many ways. Peptides including this motif bind to alternating dC-dG that has been stabilized in the Z-conformation using bromination or supercoiling, as shown by band shift assays, competition experiments, and BIAcore measurements (2Herbert A. Alfken J. Kim Y.-G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (261) Google Scholar). When linked to the nuclease domain from FokI, the resulting chimeric nuclease cuts supercoiled plasmid DNA to bracket a d(C-G)13 in the Z-conformation (3Kim Y.-G. Kim P.S. Herbert A. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12875-12879Crossref PubMed Scopus (46) Google Scholar). The protein also binds to short oligonucleotides of suitable sequence and converts them from the B- to the Z-conformation, as detected by CD and Raman spectroscopy (4Berger I. Winston W. Manoharan R. Schwartz T. Alfken J. Kim Y.-G. Lowenhaupt K. Herbert A. Rich A. Biochemistry. 1998; 37: 13313-13321Crossref PubMed Scopus (54) Google Scholar, 5Herbert A. Schade M. Lowenhaupt K. Alfken J. Schwartz T. Shylakhtenko L.S. Lyubchenko Y.L. Rich A. Nucleic Acids Res. 1998; 26: 3486-3493Crossref PubMed Scopus (80) Google Scholar). The binding of Z-DNA by Zα occurs even in the presence of a 105-fold excess of B-DNA (6Herbert A.G. Spitzner J.R. Lowenhaupt K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3339-3342Crossref PubMed Scopus (37) Google Scholar). Zαbinds poly(dC-dG), stabilized in the Z-conformation by bromination, with an equilibrium dissociation constant (K d) in the lower nanomolar range, as shown by BIAcore measurements (2Herbert A. Alfken J. Kim Y.-G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (261) Google Scholar). Although many properties of Zα have been studied, its biological function in the context of ADAR1 remains unknown. The Z-DNA binding activity of Zα was first identified in proteolytic fragments of double-stranded RNA adenosine deaminase (ADAR1) (6Herbert A.G. Spitzner J.R. Lowenhaupt K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3339-3342Crossref PubMed Scopus (37) Google Scholar) and then in the full-length enzyme (7Herbert A. Lowenhaupt K. Spitzner J. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7550-7554Crossref PubMed Scopus (120) Google Scholar). Zα has been shown to be a conserved feature of human, rat, bovine, chicken, andXenopus ADAR1 (2Herbert A. Alfken J. Kim Y.-G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (261) Google Scholar). A second related motif, Zβ, has been identified in all the ADAR1 enzymes whose sequences are known. These two motifs are separated by a linker region of conserved size; an exception is the human enzyme, in which the linker is twice as long and consists of two nearly identical copies of a module (8Patterson J.B. Samuel C.E. Mol. Cell. Biol. 1995; 15: 5376-5388Crossref PubMed Scopus (433) Google Scholar). The presence of a conserved N-terminal region containing these motifs distinguishes ADAR1 from other members of the ADAR family (9Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (430) Google Scholar, 10Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), and the N terminus has been shown to be differentially expressed (8Patterson J.B. Samuel C.E. Mol. Cell. Biol. 1995; 15: 5376-5388Crossref PubMed Scopus (433) Google Scholar). Therefore, we conclude that this region is of importance for the biological function of ADAR1. The ADAR family of enzymes converts adenosine to inosine within double-stranded regions of RNA (11Maas S. Melcher T. Seeburg P.H. Curr. Opin. Cell Biol. 1997; 9: 343-349Crossref PubMed Scopus (54) Google Scholar). In mRNA, inosine is read as guanosine by the translation apparatus, resulting in codon changes within the synthesized protein. A-to-I editing has been shown to occurin vivo in a number of mRNAs from higher animals (12Verdoorn T.A. Burnashev N. Monyer H. Seeburg P.H. Sakmann B. Science. 1991; 252: 1715-1718Crossref PubMed Scopus (678) Google Scholar, 13Hume R.I. Dingledine R. Heinemann S.F. Science. 1991; 253: 1028-1031Crossref PubMed Scopus (603) Google Scholar, 14Sommer B. Kohler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1185) Google Scholar, 15Kohler M. Burnashev N. Sakmann B. Seeburg P.H. Neuron. 1993; 10: 491-500Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 16Lomeli H. Mosbacher J. Melcher T. Hoger T. Geiger J.R. Kuner T. Monyer H. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (641) Google Scholar, 17Petschek J.P. Mermer M.J. Scheckelhoff M.R. Simone A.A. Vaughn J.C. J. Mol. Biol. 1996; 259: 885-890Crossref PubMed Scopus (44) Google Scholar, 18Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (859) Google Scholar). The best characterized of these, the editing of pre-mRNAs for subunits of the glutamate-gated cation channels in the brain, results in channels with dramatically altered functional properties (19Seeburg P.H. J. Neurochem. 1996; 66: 1-5Crossref PubMed Scopus (175) Google Scholar). Double-stranded RNA structures required for ADAR activity are formed by base pairing of an exonic sequence around the editing site with a complementary sequence in the downstream intron; therefore, editing must take place in the nucleus before splicing removes the respective intron(s). It has been proposed that Zα serves to target ADAR1 to its preferred substrates by binding to Z-DNA formed close to actively transcribing genes (20Herbert A. Rich A. J. Biol. Chem. 1996; 271: 11595-11598Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To better understand the role of Zα, we have characterized the N-terminal region of ADAR1 functionally and structurally. Using human ADAR1 as a model, the classical approach of limited proteolysis was employed to define the boundaries of this domain. Both motifs,Zα and Zβ, together are shown to form a single functional domain, Zab; Zab is stable and protected from proteolysis. Za, containing Zα, but not Zβ, can be regarded as a stable subdomain; this subdomain contributes the main binding activity. There is no equivalent subdomain containingZβ: this region is poorly structured and unstable when isolated. The intervening linker region is unexpectedly well structured, In humans, the second copy of the linker module appears to have a structure similar to the first. Removing one copy of the linker modules reduces the DNA binding affinity, indicating the importance of the distance between the Zα and Zβ motifs. Za binds Z-DNA in a conformationally specific, but not sequence-specific manner. The binding is modified by the presence of the entire Zab domain to confer preference for d(C-G)n over d(C-A)n·d(T-G)n. Different portions of the cloned cDNA coding for human ADAR1 (GenBankTM accession number U10439) were polymerase chain reaction-amplified and inserted into the expression vector pET28a (Novagen), resulting in N-terminal His6-tagged fusion proteins. In detail, Za131 (residues 96–226), Za77 (133–209), and Zab236 (133–368) were amplified using complementary primers flanked with restriction sites at their termini. Polymerase chain reaction products were analyzed on an agarose gel; bands of the correct size were extracted and subcloned into theNdeI-HindIII sites (Za131 and Za77) or theNheI-HindIII sites (Zab236) of the multiple cloning site of pET28a, resulting in the vectors pZa131, pZa77, and pZa236, respectively. Another construct, ZabΔl, missing one of the two 49-amino acid linker modules separating the Zα andZβ motifs, was created from pZab236 as follows. The 1.1-kilobase SphI-HindIII restriction fragment was digested with the restriction enzyme DrdI, resulting in two cleavage sites at identical locations at nucleotides 789 and 936 (numbers according to GenBankTM accession number U10439). The resulting DNA fragments were deproteinized and precipitated (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After incubation with T4 DNA ligase (25 °C, 4 h), the reaction mixture was analyzed on an agarose gel. The 930-base pair ligation product was isolated and subcloned in the 5-kilobaseSphI-HindIII restriction fragment of pET28a, resulting in the vector pZabΔl. To ensure that the plasmids were correct, they were analyzed by restriction digestion, and the coding regions were sequenced using Sequenase Version 2.0 (U. S. Biochemical Corp.). according to the manufacturer's instructions. The proteins were overproduced in Escherichia coli strain Novablue(DE3) (Novagen). Bacteria were grown at 37 °C in Luria-Bertani medium and induced with 1 mmisopropyl-β-d-thiogalactopyranoside at 0.7–0.9A 600 nm units. Cells were harvested after a further 3 h of growth at 37 °C. All subsequent steps were done at 4 °C. The proteins were purified essentially to homogeneity under nondenaturing conditions as follows. A cell pellet obtained from a 1-liter culture was resuspended in 15 ml of buffer A (50 mmTris-HCl (pH 8.0), 300 mm NaCl, 10 mmimidazole, 5 mm β-mercaptoethanol, 20 μg/ml RNase A, and 100 μm phenylmethylsulfonyl fluoride), and the cells were lysed using a French press. The lysate was then centrifuged for 30 min at 25,000 × g, and the clear supernatant was separated and incubated with 2 ml of Ni2+-nitrilotriacetic acid metal affinity resin (QIAGEN Inc.) for 1 h. The resin was washed three times with 20 ml of buffer A in a batch and then washed with 40 ml of buffer B (50 mm Tris-HCl (pH 8.0), 1m NaCl, 10 mm imidazole, and 5 mmβ-mercaptoethanol) in a column. Overproduced His6-tagged fusion protein was eluted with an imidazole step gradient in buffer C (50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and 5 mm β-mercaptoethanol). Steps were 30, 50, and 200 mm imidazole, respectively. Fractions were analyzed by denaturing SDS-polyacrylamide gel electrophoresis (PAGE) 1The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. on 15 or 18% gels. Fractions containing protein were pooled and dialyzed against buffer D (20 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 2 mm dithiothreitol (DTT)). After 1 h of dialysis, 15 units of thrombin (Calbiochem) were added to cleave the N-terminal His6 tag. 12 h later, the cleaved protein was dialyzed against buffer E (20 mm HEPES (pH 7.5), 20 mmNaCl, and 2 mm DTT) and finally purified by cation-exchange chromatography on a Mono S HR5/5 column (Amersham Pharmacia Biotech). Proteins were eluted with a 30-ml linear gradient of NaCl (0.05–0.3m) in 20 mm HEPES (pH 7.5) and 1 mmDTT at a flow rate of 0.7 ml/min, resulting in sharp peak profiles. Za77 eluted at 220 mm NaCl, ZabΔl at 200 mm, and Zab236 at 180 mm. The yield of electrophoretically homogeneous protein was determined using extinction coefficients of 14,000 m−1 cm−1 (Za77 and Za131), 22,400 m−1 cm−1 (ZabΔl), and 28,020 m−1 cm−1 (Zab236) at the absorbance maximum at 278 nm (calculated as described in Ref. 22Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar). 8–12 mg of protein were obtained per liter of bacterial culture. Protease digestion was performed by treating 50 μg of protein (0.5 μg/μl) with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus endoproteinase Glu-C in 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 2 mm DTT at a protein/protease mass ratio in the range of 50:1 to 1000:1 for various times at 24 °C. Reactions were stopped by heat denaturation at 100 °C for 5 min. To examine the effect of various DNA conformers on Zab digestion, the reaction was performed in 10 mm HEPES (pH 7.5), 20 mm NaCl, 5 mm DTT, and 10 mm MgCl2. DNA was used in a base pair/protein molar ratio of 5:1. Poly[d(5-MeC-G)] was used as substrate DNA, and poly[d(A-G)]·poly[d(C-T)] as unspecific DNA. The digests were separated by SDS-PAGE on 18% gels, followed by staining with Coomassie Brilliant Blue G-250. In the case of protein digested for the experiment shown in Fig. 6 (lanes 10–13), the reactions were stopped by adding phenylmethylsulfonyl fluoride (1 mm) instead of heat inactivation to ensure nondenatured protein. The proteolytic fragments were analyzed by mass spectrometry on a Voyager DE Workstation (PerSeptive) using matrix-assisted laser desorption ionization-time of flight technology. As a matrix, sinapinic acid (10 μg/μl) in acetonitrile/H2O/trifluoroacetic acid (70:29.9:0.1) was used. Alternatively, for fragments smaller than 10-kDa, the matrix was prepared with α-cyanocinnamic acid (10 μg/μl) instead of sinapinic acid. Various fragments were further analyzed by amino-terminal sequencing on an Applied Biosystems Model 475/477A protein sequencer. DNA binding was assayed by native PAGE (23Lane D. Prentki P. Chandler M. Microbiol. Rev. 1992; 56: 509-528Crossref PubMed Google Scholar). The assay was carried out using d(5-BrC-G)20 as the substrate, which is stable in the left-handed Z-DNA conformation under the applied conditions (24Malfoy B. Rousseau N. Leng M. Biochemistry. 1982; 21: 5463-5467Crossref PubMed Scopus (59) Google Scholar). The substrate was end-labeled with 32P and purified by native PAGE prior to the experiment. A reaction mixture of 10 μl containing the ADAR1 fragment (4–500 nm) with <1 pm substrate in 10 mm Tris-HCl (pH 7.8), 20 mm NaCl, 5 mm DTT, 5% glycerol, 100 μg/ml bovine serum albumin, and 50 μg/ml poly[d(A-G)]·poly[d(C-T)] (Amersham Pharmacia Biotech) as an unspecific competitor was incubated for 30 min at 24 °C. The mixture was analyzed on a 6% native polyacrylamide gel using 0.5× Tris borate (22.5 mm) as the running buffer. After electrophoresis (10 V/cm, 90 min), the gel was dried and autoradiographed at –70 °C on Kodak X-Omat Blue film with intensifying screens. CD spectra were recorded at 24 °C on an Aviv Model 62DS spectrometer. Conformational change in DNA oligomers was monitored between 235 and 305 nm. DNA samples used were annealed prior to the experiment. For this purpose, a concentrated solution of the self-complementary sequence d(C-G)6 or an equimolar amount of d(C-A)7 and d(T-G)7 was heated to 85 °C for 10 min and then slowly cooled to <20 °C over 1 h. Measurements were carried out in 10 mm sodium phosphate (pH 7.0), 10 mm NaF, 1 mm EDTA, and 2 mm DTT using a DNA concentration of 30.0 μmbase pairs and an optical path length of 5 mm. Spectra were recorded in 10-nm steps and averaged over 4 s. Protein was added to the sample from a concentrated stock solution, in aliquots never exceeding 5% of the total volume, and the mixture was equilibrated for 5 min before each measurement. The spectra were corrected for buffer base line and smoothed using software provided by Aviv. Protein spectra were recorded between 190 and 250 nm. Za77 was measured at a concentration of 10.0 μm, and ZabΔl and Zab were measured at 5.0 μm and an optical path length of 1 mm. Spectra were measured in 1-nm steps and averaged over 10 s. For comparisons of the spectra of Zab between 190 and 250 nm in the presence and absence of substrate, poly[d(5-MeC-G)] was used as substrate. A 2:1 base pair/protein molar ratio was used. Protein domains are usually well structured regions of 50–200 amino acids (25Janin C. Chothia C. Methods Enzymol. 1985; 115: 420-430Crossref PubMed Scopus (64) Google Scholar, 26Schulz G. Schirmer R. Principles of Protein Structure. Springer-Verlag New York Inc., New York1979Crossref Google Scholar). Larger proteins are built from multiple, mostly independently folded domains. The regions connecting those domains are often flexible and solvent-exposed. Limited proteolysis is a classical approach to define domain organization (27Porter R. Science. 1973; 180: 713-716Crossref PubMed Scopus (99) Google Scholar, 28Jovin T. Geisler N. Weber K. Nature. 1977; 269: 668-672Crossref PubMed Scopus (52) Google Scholar, 29Roy R. Kumar A. Lee J.C. Mitra S. J. Biol. Chem. 1996; 271: 23690-23697Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 30Nakagawa N. Masui R. Kato R. Kuramitsu S. J. Biol. Chem. 1997; 272: 22703-22713Crossref PubMed Scopus (24) Google Scholar). It takes advantage of the fact that site-specific proteases will cleave proteins preferentially in solvent-exposed unstructured regions, rather than within a folded domain. Limited proteolysis was used to define a structured core containingZα, the Z-DNA-binding motif present in the N-terminal region of ADAR1. Zα has been defined to comprise residues 121–197 of human ADAR1 using functional assays (2Herbert A. Alfken J. Kim Y.-G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (261) Google Scholar). However, a variety of results from nondenaturing electrophoresis, chromatographic elution, and NMR studies have suggested that the recombinantly produced peptide is not stably folded. 2K. Lowenhaupt and T. Schwartz, unpublished results., 3M. Schade and I. Berger, personal communication. An N- and C-terminally extended portion of the ADAR1 N terminus comprising Gly96–Ser226 was overproduced as a His6-tagged fusion protein in E. coli, and its digestion with four different proteases (endoproteinase Glu-C, chymotrypsin, thermolysin, and trypsin) was analyzed. Each of these enzymes has a different sequence specificity; therefore, using them in concert results in complementary information. The use of this combination of proteases results in an even distribution of potential cleavage sites throughout the studied protein, with gaps no longer than 4 residues between adjacent sites. A time course of cleavage with endoproteinase Glu-C is shown in Fig.1 A. An 11-kDa band appeared rapidly and increased in intensity over the observed time; the full-length protein band gradually disappeared over the same period. The intensity of the 11-kDa band was comparable to that of the full-length band, indicating a stoichiometric conversion to a stable product. The cleavage site was mapped to a preferential endoproteinase Glu-C site, C-terminal to Asp132, using N-terminal sequencing. Similar results were obtained using trypsin and chymotrypsin to cleave this protein (data not shown). To ensure that a minimum domain had been identified, the protein was cleaved sequentially with two different proteases. Fig. 1 Bshows the digestion with endoproteinase Glu-C followed by chymotrypsin. Before addition of the second protease, only the 11-kDa band was detectable. Chymotrypsin further truncated the fragment, producing the stable product V8/Ch-8. The N and C termini of these fragments were identified unambiguously using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The V8–11 fragment was shown to contain residues 133–226. Chymotrypsin cut after Trp204; V8/Ch-8 consists of residues 133–204. A similar digestion, carried out with endoproteinase Glu-C and thermolysin, produced a stable product extending from amino acids 133 to 209 (data not shown). Other combinations of enzymes produced consistent results in all cases. From this, we conclude that there is a core domain containing Zα. Trp204 is a potential target for cleavage by both chymotrypsin and thermolysin. Chymotrypsin cut well, but thermolysin cut only marginally at Trp204. Therefore, we define the core domain as comprising Leu133–Gly209. This core was in no case significantly degraded, whereas the regions on either end were rapidly degraded to pieces too small to detect. These results were used to design a stable construct, Za, comprising Leu133–Gly209. This protein was purified fromE. coli undegraded under nondenaturing conditions. Za showed superior chromatographic behavior over previous Zαconstructs, purifying from a Mono S cation-exchange column homogeneously as a sharp peak; this indicated structural uniformity. Samples yielded a single band when analyzed by native PAGE. 4T. Schwartz, unpublished results. When challenged with exogenous proteases, only Za showed striking stability; otherZα constructs were rapidly degraded (data not shown). Both Zα and Zβ are present in every species in which the sequence of ADAR1 is known. The motifs are separated by one or two copies of a module, weakly conserved in sequence, but consistently lacking positively charged residues and 43–49 amino acids in length. 12 residues from this module are an essential part of Za, the stable Zα core domain. It seemed possible that Zα, Zβ, and the linker module(s) together form a single structural and functional unit. To investigate this possibility, we examined the structural organization of a peptide spanning both DNA-binding motifs. This peptide, termed Zab, comprising Leu133 (the previously defined N terminus of Za) to Asn368 (C-terminal to Zβ, from human ADAR1), was soluble when overproduced in E. coli, and full-length protein could be obtained with high yield. These results indicate proper folding with no significant instability. Improper folding often leads to the formation of inclusion bodies inside the overproducing bacterial cell (31Wilkinson D.L. Harrison R.G. Bio/Technology. 1991; 9: 443-448Crossref PubMed Scopus (106) Google Scholar). Highly flexible proteins are frequently degraded if expressed in a foreign host (32Makrides S.C. Microbiol. Rev. 1996; 60: 512-538Crossref PubMed Google Scholar). The results of the digestion of Zab with four different proteases are shown in Fig. 2. Each enzyme cleaved in a characteristic pattern and produced a small number of very stable bands. Time points were selected to allow the identification of all stable products, using mass spectrometry and N-terminal sequencing where appropriate; minor products were identified wherever possible. In each case, well resolved spectra were recorded. TableI lists the peptides produced by each enzyme, as determined from the molecular mass. For chymotrypsin, trypsin, and endoproteinase Glu-C, the assignments are unambiguous and in good agreement with SDS-PAGE analysis. Minor exceptions are fragments Tr8 and Ch5, which were detected only by mass spectrometry, as discussed below. In the case of thermolysin, it was not possible to unambiguously assign the multiple transitory fragments; however, the major fragments seen after 60 min of digestion could be identified. A schematic diagram of the major transitory products and the stable proteolytic fragments is shown in Fig.3.Table IMass spectroscopic analysis of Zab236 fragmentsProteolytic fragmentMeasured massSequence assignmentCalculated massDaDaV8V2625,650133L . . . E3611-aFragment contains 6 additional N-terminal vector-encoded residues.25,666V2019,756133L . . . L3071-aFragment contains 6 additional N-terminal vector-encoded residues.19,716V1918,841133L . . . K3001-aFragment contains 6 additional N-terminal vector-encoded residues.18,850V1413,536240D . . . E36113,539V1212,173133L . . . E2391-aFragment contains 6 additional N-terminal vector-encoded residues.12,126ChymotrypsinCh1818,082205N . . . N36818,119Ch1312,918254N . . . N36812,907Ch87865135I . . . W2047851Ch55189205N . . . W2535230ThermolysinTl1212,239261R . . . M36312,186Tl88495134S . . . G2098460TrypsinTr1919,103133L . . . K3021-aFragment contains 6 additional N-terminal vector-encoded residues.19,102Tr1515,309233N . . . N36815,310Tr1211,600133L . . . R2321-aFragment contains 6 additional N-terminal vector-encoded residues.11,587Tr87559233N . . . K30275391-a Fragment contains 6 additional N-terminal vector-encoded residues. Open table in a new tab Figure 3Structure and protease cleavage map of the Z-DNA-binding domain of ADAR1. At the top is a schematic representation of human ADAR1 (hADAR1). Below are the stable fragments produced by limited proteolysis. Numbers above ADAR1 are residue positions. The illustrations are proportional.dsRNA, double-stranded RNA.View Large Image Figure ViewerDownload (PPT) Endoproteinase Glu-C cleaved Zab rapidly at a single site, Glu361 at the extreme C terminus (Fig. 2 A). The resulting peptide was very stable to further proteolysis, despite an abundance of potential cleavage sites, including Asp132, which is exquisitely sensitive in the shorter construct used to define Za, as described above. After a long incubation with large amounts of enzyme, additional cleavage occurred at Glu239, Glu301, and Leu307. Glu239 lies within the first 49-amino acid repeat; remarkably, the equivalent site in the second repeat, Glu288, was uncut. Chymotrypsin cleaved the protein after Trp204 and Trp253. These sites are at equivalent positions in the two copies of the tandem repeat. The three generated fragments were stable (Fig. 2 B). The 5-kDa fragment was not visible on SDS-polyacrylamide gel, although it generated a signal in mass spectrometry of comparable intensity to Ch12 and Ch8. Coomassie Blue staining depends largely on positive charges present in the peptide (33Tal M. Silberstein A. Nusser E. J. Biol. Chem. 1985; 260: 9976-9980Abstract Full Text PDF PubMed Google Scholar). The 49-amino acid repeat contains only 1 positively charged residue. Therefore, we speculated that although Ch5 was resolved on the gel, it was not stained. Two other transitory fragments, Ch18 and Ch*, were separated on the gel. Ch18 could be assigned to be the product of a single cutting site. Ch* could not be unambiguously determined by mass spectrometry. Thermolysin produced similar stable products (Fig. 2 C). Again, symmetrical sites in the repeated linker, Gly209 and Gly258, were cut, resulting in two stable products on an SDS-polyacrylamide gel. Because thermolysin has low sequence specificity, many transitory products were seen, especially at early time points. Because of these products, it was not possible to unambiguously identify the Tl5 fragment from among several candidates seen by mass spectrometry. Trypsin attacked the protein at two preferred sites, Arg232in the firs