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• W2073161229 abstract "The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241–12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)15 using two independent methods. All mutations in the middle of HCDR3 led to either abolished or diminished affinity. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge. The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241–12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)15 using two independent methods. All mutations in the middle of HCDR3 led to either abolished or diminished affinity. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge. Antibodies comprise a valuable class of protein produced in mammals and other species in response to antigen. The importance of antibodies is highlighted by their vital roles in immune protection. Mammalian immune systems are capable of generating millions of different antibody specificities in response to foreign antigens. For unknown reasons, a breakdown in immune tolerance can occur resulting in activation of self-reactive B and T cells. This abnormal cascade of events leads to autoimmune disease. A hallmark of the autoimmune disorders systemic lupus erythematosus (SLE) 1The abbreviations used are: SLE, systemic lupus erythematosus; ds, double-stranded; Fab, antigen binding fragment(s); ss, single-stranded; MRL/lpr, MRL/MpJ-lpr/lpr; H, heavy; L, light; CDR, complementarity-determining region(s); NTA, nitrilotriacetic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TNM, tetranitromethane; Fv, variable region; ΔΔG°, ΔG°mutant − ΔG°DNA-1. and mixed connective tissue disease is the presence of serum antibodies that recognize nucleic acids (1Tan E.M. Adv. Immunol. 1989; 44: 93-152Crossref PubMed Scopus (1381) Google Scholar, 2Stollar B.D. CRC Crit. Rev. Biochem. 1986; 21: 1-36Crossref PubMed Scopus (54) Google Scholar, 3Hoffman R.W. Sharp G.C. Deutscher S.L. Arthritis Rheum. 1995; 38: 1837-1844Crossref PubMed Scopus (51) Google Scholar). The presence of high levels of circulating antibodies that bind double-stranded (ds) DNA is diagnostic for SLE, and certain anti-DNA immune complexes contribute to disease pathology (4Pisetsky D.S. Grudier J.P. Gilkeson G.S. Arthritis Rheum. 1990; 33: 153-159Crossref PubMed Scopus (97) Google Scholar, 5Stollar B.D. FASEB J. 1994; 8: 337-342Crossref PubMed Scopus (64) Google Scholar). It is uncertain what are the distinguishing features of pathogenic versus non-pathogenic antibodies, however. Studies of the binding properties of anti-DNA antibodies have been facilitated by methods to select for DNA-binding antibody fragments (Fab) from bacteriophage display libraries and the ability to produce large quantities of the Fab in Escherichia coli. We previously isolated an anti-single-stranded (ss) DNA-binding Fab, DNA-1, from a bacteriophage display library derived from the immunoglobulin repertoire of an autoimmune MRL/MpJ-lpr/lpr (MRL/lpr) mouse (6Calcutt M.J. Kremer M.T. Giblin M.F. Quinn T.P. Deutscher S.L. Gene (Amst.). 1993; 137: 77-83Crossref PubMed Scopus (21) Google Scholar). The MRL/lpr murine library was enriched in anti-DNA Fab since these mice spontaneously produce anti-ssDNA and anti-dsDNA antibodies and develop an SLE-like syndrome. DNA-1 was shown to preferentially bind to oligo(dT) of 15 nucleotides or greater in length, with an equilibrium dissociation constant (K d) of 150–200 nm (7Calcutt M.J. Komissarov A.A. Marchbank M.T. Deutscher S.L. Gene (Amst.). 1996; 168: 9-14Crossref PubMed Scopus (12) Google Scholar, 8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The regions responsible for DNA binding are known to reside in the three heavy (H) and light (L) chain complementarity-determining regions (CDRs) of the Fab. It has been shown that the H chain contributes more to the interaction with DNA than the L chain (9Kabat E.A. Padlan A. Proc. Natl. Acad. Sci. U. S. A. 1989; 85: 6885-6889Google Scholar). Comparison of HCDR transplantation mutants between DNA-1 and a Fab that bound poorly to DNA demonstrated that HCDR3 of DNA-1 was critical for oligo(dT) binding (7Calcutt M.J. Komissarov A.A. Marchbank M.T. Deutscher S.L. Gene (Amst.). 1996; 168: 9-14Crossref PubMed Scopus (12) Google Scholar, 8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The importance of HCDR3 in binding to other DNA molecules including Z-DNA (10Polymenis M. Stollar B.D. J. Immunol. 1995; 156: 3065-3073Google Scholar), dsDNA (11Barbas S.M. Ditzel H.J. Salonen E.M. Yang W. Silverman G.J. Burton D.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2529-2533Crossref PubMed Scopus (126) Google Scholar, 12Pewzner-Jung Y. Simon T. Eilat D. J. Immunol. 1996; 154: 2198-2208Google Scholar), and ssDNA (13Gulliver G.A. Bedzyk W.D. Smith R.G. Bode S.L. Tetin S.Y. Voss Jr., E.W. J. Biol. Chem. 1994; 269: 7934-7940Abstract Full Text PDF PubMed Google Scholar, 14Gulliver G.A. Rumbley C.A. Carrero J. Voss Jr., E.W. Biochemistry. 1995; 34: 5158-5163Crossref PubMed Scopus (10) Google Scholar) has been demonstrated. Previous data generated from x-ray analyses of anti-protein antibodies indicated that HCDR3 does not participate in secondary structure formation and represents a loop, often with a putative salt bridge at its base (15Chothia C. Lesk A.M. J. Mol. Biol. 1987; 196: 901-917Crossref PubMed Scopus (1191) Google Scholar) that may function to stabilize loop conformation. That HCDR3 loop residues are critical in antigen recognition has been demonstrated in a limited number of studies (16Rumbley C.A. Denzin L.K. Yantz L. Tetin S.Yu. Voss Jr., E.W. J. Biol. Chem. 1993; 268: 13667-13674Abstract Full Text PDF PubMed Google Scholar, 17Brummell D.A. Sharma V.P. Anand N.N. Bilous D. Dubic G. Michniewicz J. MacKenzie C.R. Sadowska J. Sigurskjold B.W. Sinnott B. Young N.M. Bundle D.R. Narang S.A. Biochemistry. 1993; 32: 1180-1187Crossref PubMed Scopus (70) Google Scholar, 18Jackson J.R. Sathe G. Rosenberg M. Sweet R. J. Immunol. 1995; 154: 3310-3319PubMed Google Scholar). Nevertheless, the precise function of HCDR3 in DNA interaction has not been elucidated. In the present study, the role of amino acid residues of HCDR3 of DNA-1 in oligo(dT) interaction was examined. Two regions were chosen for mutagenesis experiments based on a molecular model of DNA-1: (i) residues in the middle of HCDR3 (Tyr101-Arg-Pro-Tyr-Tyr105), and (ii) the amino acids Arg98 and Asp108 at the base of the loop, which have been proposed to form a salt bridge. Both fluorescence quenching titration (8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and a Ni-NTA-agarose radioimmunoassay (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar) were used for comparison of the affinities of 25 purified HCDR3 mutants of DNA-1. The data obtained highlighted the critical role of tyrosine residues in the central portion of HCDR3 in proper Fab-oligonucleotide complex formation. A change in the size as well as an increase in the negative charge of the HCDR3 loop resulted in a loss of ssDNA binding, while elimination of the sole carboxylic group resulted in increased affinity for (dT)15. Gamma-bind Sepharose, Sephadex G-50M, and a Mono-S column were purchased from Pharmacia Biotech Inc. Ni-NTA-agarose was purchased from Qiagen Corp. (Chatsworth, CA). [γ-32P]ATP (3000 Ci/mol) was obtained from NEN Life Science Products. Protein molecular weight standards were from Novex (San Diego, CA). All other reagents were obtained from Sigma or Fisher unless otherwise noted. The (dT)15 was synthesized by the University of Missouri DNA Core Facility, using an Applied Biosystems DNA synthesizer (model 380B), reverse-phase column purified and quantitated spectrophotometrically (20Kowalczykowski S.C. Lonberg N. Newport J.W. von Hippel P.H. J. Mol. Biol. 1981; 145: 75-104Crossref PubMed Scopus (259) Google Scholar), and terminally phosphorylated with [γ-32P]ATP by T4 polynucleotide kinase. Unincorporated radioactivity was removed by separation on Chroma Spin-10 columns (CLONTECH). The framework region of DNA-1 was modeled on the crystal structure of the mouse anti-hapten antibody R19.9 because of the high sequence homology with DNA-1 and because five out of six CDRs were identical in length to their counterparts (21Lascombe M.B. Alzari P.M. Boulot G. Saludjian P. Tougard P. Berek C. Haba S. Rosen E.M. Nisonoff A. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 607-611Crossref PubMed Scopus (84) Google Scholar). The DNA-1 sequence was aligned with the R19.9 sequence and then substituted into the R19.9 structure using the biopolymer and homology modeling tools present in SYBYL (Tripos, Inc). Once the framework of DNA-1 was completed, the CDRs were added to the model. The loop structure of the HCDR3 from DNA-1, which differed in sequence length from that of antibody 19.9 by an additional two residues, was generated from the x-ray structure of HCDR3 of an anti-p185HER2 antibody of similar sequence and identical length (22Eigenbrot C. Randal M. Presta L. Carter P. Kossiakoff A.A. J. Mol. Biol. 1993; 229: 969-995Crossref PubMed Scopus (180) Google Scholar). In accordance with the molecular model of the combining site (Fig. 1 A), HCDR3 represents a loop with the potential for formation of a salt bridge at its base as a result of the interaction of a conserved Arg98 residue in framework region 3 and Asp108 at the C terminus of HCDR3 (Fig.1 B). Results of x-ray analysis together with data from site-directed mutagenesis of different antibodies (15Chothia C. Lesk A.M. J. Mol. Biol. 1987; 196: 901-917Crossref PubMed Scopus (1191) Google Scholar, 16Rumbley C.A. Denzin L.K. Yantz L. Tetin S.Yu. Voss Jr., E.W. J. Biol. Chem. 1993; 268: 13667-13674Abstract Full Text PDF PubMed Google Scholar) suggest that residues in the central portion of the HCDR3 loop likely participate in direct interactions with the antigen at the combining site, while residues placed near the base of HCDR3 are more likely to contribute to the conformational support or flexibility of the loop. Therefore, the five residues in the middle of HCDR3 (Tyr101-Arg-Pro-Tyr-Tyr105) and two residues at the base of the loop (Arg98 and Asp108) were chosen for site-directed mutagenesis experiments to examine the possible contributions of the separate amino acid residues in complex formation. The mutants generated are shown in Table I.Table IList of HCDR3 mutants generatedFabFR31-aFR, framework region.HCDR3DNA-11-bHCDR3, mutated residues in bold.98RGGYRPYYAMDY109HCDR3 middle Y101F---F-------- Y101H---H-------- Y101S---S-------- Y101N---N-------- Y101D---D-------- Y101C---C-------- R102K----K------- R102M----M------- R102T----T------- −P1031-cDeletion mutation.----------- P103G-----G------ P103T-----T------ P103C-----C------ P103R-----R------ R102A/P103R1-dDouble mutation.----AR------ P103AA1-eInsertion mutation.-----AA------ Y104F------F----- Y104H------H----- Y104D------D----- Y105F-------F---- Y105F/A106V1-dDouble mutation.-------FV--- Y105C-------C----Putative salt bridge R98AA----------- D108A----------A- R98A/D108A1-dDouble mutation.A---------A-1-a FR, framework region.1-b HCDR3, mutated residues in bold.1-c Deletion mutation.1-d Double mutation.1-e Insertion mutation. Open table in a new tab VSX, a derivative of the plasmid pBC (Stratagene) containing a chloramphenicol resistance gene and lacking the VspI and SacI restriction enzyme sites, was used as an intermediate vector for mutagenesis and sequencing procedures. Site-directed mutagenesis was performed using splice overlap polymerase chain reaction (PCR) (23Higuchi R. PCR Protocols: A Guide to Methods and Applications. Academic Press, New York1990Google Scholar). Typically, two fragments of DNA-1 H chain DNA in VSX were amplified in two separate reactions using standard PCR conditions, resulting in fragments overlapping by at least 10 nucleotides. Full-length variable H + constant H1 products were generated by annealing and extension of the two overlapping fragments, and were amplified by 25 cycles of PCR following the addition of flanking T3 and T7 primers. The final products were purified, cut with XhoI andVspI restriction enzymes, and ligated into similarly cut DNA-1-VSX plasmid. Having verified that the desired mutation had been introduced, an XhoI-BamHI fragment containing the variable H region was subcloned intoXhoI-BamHI-cut DNA-1-pComb3, thus replacing the wild-type sequence. Using this two-step procedure, only a 140-nucleotide fragment derived from PCR was present in the final construct, thus reducing the amount of DNA sequencing necessary to check for possible mutations caused by Taq DNA polymerase errors. The correct mutation was verified by DNA sequencing using a Sequenase 2.0 kit (U. S. Biochemical Corp.). The recombinant H and L chain DNA-1 and HCDR genes were cloned into pComb3/6-His (24Collet T.A. Roben P. O'Kennedy R.O. Barbas C.F. Burton D.R. Lerner R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10026-10030Crossref PubMed Scopus (82) Google Scholar), expressed in E. coli DH12S, and purified as described (7Calcutt M.J. Komissarov A.A. Marchbank M.T. Deutscher S.L. Gene (Amst.). 1996; 168: 9-14Crossref PubMed Scopus (12) Google Scholar). The Fab preparations were more than 957 pure, as evidenced by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar), and had aA 280/A 260 ratio > 1.90. Protein concentration was determined by a bicinchoninic acid method (26Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar) using bovine serum albumin as a standard. DNA-1 was modified with TNM as described (27Sokolovsky M. Riordan J.F. Vallee B.L. Biochemistry. 1966; 5: 3582-3589Crossref PubMed Scopus (519) Google Scholar). Portions (10 ॖl) of a 100 mm stock solution of TNM in acetonitrile were added to 1.0 ml of DNA-1 (20 ॖm) in 0.1 m Tris/HCl buffer, pH 8.0, and the mixture was incubated at room temperature. After incubation for 30 min, excess reagent was removed by gel filtration on a Sephadex G-50M column. The number of modified tyrosine residues was estimated spectrophotometrically using a value of 2750m−1·cm−1 for the extinction coefficient of nitrotyrosine at 360 nm (27Sokolovsky M. Riordan J.F. Vallee B.L. Biochemistry. 1966; 5: 3582-3589Crossref PubMed Scopus (519) Google Scholar). To examine the ability of HCDR3 mutants of DNA-1 to bind oligonucleotide, a Ni-NTA microtiter plate radioimmunoprecipitation assay was utilized (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar). Mixtures (200 ॖl) of 32P-labeled (dT)15 in 0.05 m Tris/HCl, pH 7.0, with 0.1m NaCl were incubated with increasing concentrations of Fab (5–2000 nm) in the wells of microtiter plates at room temperature for 15 min until equilibrium was established. To separate bound oligonucleotide from unbound, a 40-ॖl suspension of Ni-NTA-agarose was added to each mixture to trap the Fab-32P(dT)15 complex through the hexahistidine tag at the C terminus of the H chain (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar). Immobilized complex was quantitatively eluted with 400 mm imidazole. All radioactive samples were counted using a Tri-Carb 2100 TR automatic liquid-scintillation analyzer (Packard Instrument Co., Downers Grove, IL) for 2 min using an automatic 32P window. Changes in protein fluorescence at 346 nm (292 nm excitation) over the course of Fab-(dT)15 complex formation were measured as described previously (8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The titrations were performed with varying amounts of oligonucleotide (5–20,000 nm) added to a fixed Fab concentration (10–60 nm) in 2 ml of 0.05 mTris/HCl, pH 7.0, with 0.1 m NaCl. Fluorescence titration experiments were carried out using a SLM 8100 spectrofluorimeter interfaced to a Dell/433 PC running SLM AMINCO 8100 series 2 software. The temperature of the cell compartment (25 °C) was controlled using a constant temperature cell holder connected to a circulating water bath, RTE-111 (NESLAB, Portsmouth, NH). The K d values for Fab-oligonucleotide complexes were determined from binding isotherms using a single binding site curve-fitting procedure as described previously (8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Our previous results (7Calcutt M.J. Komissarov A.A. Marchbank M.T. Deutscher S.L. Gene (Amst.). 1996; 168: 9-14Crossref PubMed Scopus (12) Google Scholar, 8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) demonstrated that HCDR3 plays a principal role in the interaction of DNA-1 and oligo(dT). In the present study, HCDR3 site-specific mutants were generated based on a molecular model of DNA-1 to better define amino acid residues important in ssDNA interaction and their possible contribution to the mechanism of antigen recognition. A molecular model for the DNA-1 variable region (Fv) was constructed to assist in the visualization of the amino acids located in the antigen combining site and to integrate experimental data with subsequent interpretation (Fig.1 A). Two regions of HCDR3 were the primary focus for directed mutagenesis experiments: the middle of HCDR3, which was predicted to participate in direct interaction with antigen, and residues Arg98 and Asp108 at the base of the loop, which may form a putative salt bridge to maintain the conformation of HCDR3 (Fig. 1 B) (28Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (794) Google Scholar). As shown in Table I, three of the five residues in the middle of HCDR3 of DNA-1 are tyrosines. Antibody combining sites are often rich in aromatic residues (28Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (794) Google Scholar, 29Padlan E.A. Proteins Struct. Funct. Genet. 1990; 7: 112-124Crossref PubMed Scopus (228) Google Scholar, 30Mian I.S. Bradwell A.R. Olson A.J. J. Mol. Biol. 1991; 217: 133-151Crossref PubMed Scopus (393) Google Scholar) that have been shown to be important for the interaction with antigen (14Gulliver G.A. Rumbley C.A. Carrero J. Voss Jr., E.W. Biochemistry. 1995; 34: 5158-5163Crossref PubMed Scopus (10) Google Scholar,31Herron J.N. He X.M. Mason M.L. Voss Jr., E.W. Edmundson A.B. Proteins Struct. Funct. Genet. 1989; 5: 271-280Crossref PubMed Scopus (213) Google Scholar, 32Cygler M. Rose D.R. Bundle D.R. Science. 1991; 253: 442-445Crossref PubMed Scopus (247) Google Scholar, 33Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Possible mechanisms for antigen interaction include formation of hydrogen bonds by the phenolic hydroxyl group and aromatic-aromatic or hydrophobic interactions (33Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Therefore, each tyrosine studied was first changed to a phenylalanine (Fab mutants Y101F, Y104F, and Y105F) to detect the possible involvement of the phenolic hydroxyl in hydrogen bonding (Table I). Mutation of Tyr101 and Tyr104 to His (Fab mutants Y101H and Y104H), was created to test the effects of changing the phenolic ring to an imidazole, on Fab affinity. Mutants Y101D, Y104D, Y101C, and Y105C were produced to examine the influence of addition of a negatively charged residue or group with reduced pK a to the top of HCDR3. In addition, two point mutants in position 101 (Y101S and Y101N) were created to explore the possible role of size and hydrophobicity of the side chain on ssDNA binding. The double mutant Y105F/A106V was generated to examine the influence of an increase in potential for hydrophobic interactions in this region of the HCDR3 loop. In accordance with the molecular model of DNA-1, HCDR3 was predicted to form a reverse turn at Pro103 (Fig. 1). Four substitution mutants (P103G, P103T, P103C, and P103R) were produced to explore the influence of the change in size and charge at this position on ssDNA affinity. Insertion and deletion mutations (mutant Fab −P103 and P103AA) were also created to study the role of the size of HCDR3 in the interaction with oligo(dT) (Table I). Combining sites of anti-DNA antibodies often contain arginines and lysines that can interact with the negatively charged phosphates of the antigen (2Stollar B.D. CRC Crit. Rev. Biochem. 1986; 21: 1-36Crossref PubMed Scopus (54) Google Scholar, 5Stollar B.D. FASEB J. 1994; 8: 337-342Crossref PubMed Scopus (64) Google Scholar, 10Polymenis M. Stollar B.D. J. Immunol. 1995; 156: 3065-3073Google Scholar, 34Marion T.N. Tillman D.M. Jou N.T. Hill R.J. Immunol. Rev. 1992; 128: 123-149Crossref PubMed Scopus (110) Google Scholar, 35Eilat D. Anderson W.F. Mol. Immunol. 1994; 31: 1377-1390Crossref PubMed Scopus (68) Google Scholar, 36Herron J.N. He X.M. Ballard D.W. Blier P.R. Pace P.E. Bothwell A.L.M. Voss Jr., E.W. Edmundson A.B. Proteins Struct. Funct. Genet. 1991; 11: 159-175Crossref PubMed Scopus (272) Google Scholar, 37Radic M.Z. Weigert M. Annu. Rev. Immunol. 1994; 12: 487-520Crossref PubMed Scopus (432) Google Scholar). Three mutant variants with replacement of Arg102 (R102K, R102M, and R102T) and double mutant R102A/P103R were generated in an effort to identify the possible role of the positive charge at the top of HCDR3 loop and the effect of the shift of the arginine residue on the Fab-ssDNA complex formation, respectively (Table I). Finally, the role of the Arg98 and Asp108residues in supporting the functional integrity of the DNA-1 combining site was examined by generation of a 舠double mutant cycle舡 (38Carter P.J. Winter G. Wilkinson A.J. Fersht A.R. Cell. 1984; 38: 835-840Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 39Ackers G.K. Smith F.R. Annu. Rev. Biochem. 1985; 4: 597-629Crossref Scopus (197) Google Scholar, 40Horovitz A. J. Mol. Biol. 1987; 196: 733-735Crossref PubMed Scopus (82) Google Scholar) consisting of two point mutants and one double mutant (R98A, D108A, and R98A/D108A, respectively). Taking advantage of the principle of additivity (38Carter P.J. Winter G. Wilkinson A.J. Fersht A.R. Cell. 1984; 38: 835-840Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 39Ackers G.K. Smith F.R. Annu. Rev. Biochem. 1985; 4: 597-629Crossref Scopus (197) Google Scholar, 40Horovitz A. J. Mol. Biol. 1987; 196: 733-735Crossref PubMed Scopus (82) Google Scholar), the comparison of the changes in ΔG° for point mutations with the values obtained for the double mutant and wild type Fab may indicate the possible interaction between Arg98 and Asp108 and its role in formation of the Fab-ligand complex. Individual HCDR3 site-directed mutant versions of DNA-1 were produced in E. coli DH12S (8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and purified to homogeneity using affinity and ion-exchange chromatography (Fig.2 A) as described under 舠Experimental Procedures.舡 Results of SDS-PAGE analysis of the Fab employed in this study are shown in Fig. 2 (B andC). Samples of DNA-1 and all 25 HCDR3 mutants purified demonstrated neither a decrease in affinity or proteolysis after 6 months of storage at 4 °C. Changes in fluorescence emission induced by denaturation of Fab after incubation for 15 min at 35–60 °C (41Deutscher S.L. Crider M.E. Ringbauer J. Komissarov A.A. Quinn T.P. Arch. Biochem. Biophys. 1996; 333: 207-213Crossref PubMed Scopus (12) Google Scholar) were used as an indicator of the influence of HCDR3 alterations on the overall structure of the molecule. Using this method, the thermostability of each mutant studied was similar to DNA-1 (data not shown). These results were in agreement with the supposition that HCDR3 plays a negligible role in the support of Fab structure. Ni-NTA precipitation of Fab-oligonucleotide complexes (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar) was employed for the rapid and facile analysis of the binding properties of HCDR3 mutants and for the direct estimation ofK d values. All Fab studied contained a hexahistidine tag at the C terminus of their H chain (24Collet T.A. Roben P. O'Kennedy R.O. Barbas C.F. Burton D.R. Lerner R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10026-10030Crossref PubMed Scopus (82) Google Scholar), which did not interfere with oligonucleotide interaction (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar, 41Deutscher S.L. Crider M.E. Ringbauer J. Komissarov A.A. Quinn T.P. Arch. Biochem. Biophys. 1996; 333: 207-213Crossref PubMed Scopus (12) Google Scholar). Since previous studies have shown that DNA-1 exhibits preferential specificity for oligo(dT) and has maximal affinity for a ligand 15 or more bases in length (7Calcutt M.J. Komissarov A.A. Marchbank M.T. Deutscher S.L. Gene (Amst.). 1996; 168: 9-14Crossref PubMed Scopus (12) Google Scholar, 8Komissarov A.A. Calcutt M.J. Marchbank M.T. Peletskaya E.N. Deutscher S.L. J. Biol. Chem. 1996; 271: 12241-12246Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), all binding experiments were performed with (dT)15. The Fab·32P-(dT)15 complexes were separated from unbound ligand by precipitation with Ni-NTA-agarose and quantitatively eluted with imidazole (19Komissarov A.A. Marchbank M.T. Deutscher S.L. Anal. Biochem. 1997; 247: 123-129Crossref PubMed Scopus (6) Google Scholar). The relative affinities of the Fab (500 nm) to (dT)15 (5 nm) are presented in Fig. 3. As shown, modification of DNA-1 with TNM and 11 of 25 HCDR3 mutations in DNA-1 resulted in a dramatic decrease in the ability of the Fab to bind (dT)15. All alterations that resulted in a significant decrease in the pK a, accompanied with an increase in the negative charge of the residue (Y101D, Y104D, Y101C, Y105C, P103C, modification with TNM), led to a significant decrease in binding ability. Mutants with a change in the length of HCDR3 (−P103 and P103AA) as well as both double mutants directed to the middle of the loop (R102A/P103R and Y105F/A106V) were also greatly diminished in their oligo(dT) binding ability. Therefore, neither the appearance of negatively charged groups (or groups possessing a lower pK a than tyrosine) at the top of HCDR3 nor alterations in the size of the loop were tolerated without a significant reduction in binding. The estimated affinities from titrations of 32P-(dT)15 with Fab are shown in Tables II andIII. These results demonstrated that any alteration to the top of HCDR3 resulted in decreased affinity, underscoring the importance of this region in ligand interaction (TableII)" @default.
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