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1.
Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops. 相似文献
2.
Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition 总被引:2,自引:0,他引:2
Schuermann JP Prewitt SP Davies C Deutscher SL Tanner JJ 《Journal of molecular biology》2005,347(5):965-978
Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity. 相似文献
3.
Yokoyama H Mizutani R Satow Y Komatsu Y Ohtsuka E Nikaido O 《Journal of molecular biology》2000,299(3):711-723
DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues. 相似文献
4.
Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG) 总被引:1,自引:0,他引:1
Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed. 相似文献
5.
11F8 is a pathogenic monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse. Previous studies established that 11F8 is sequence-specific and identified the thermodynamic and kinetic basis for the specific recognition of ssDNA, and binding site mutations of a single-chain construct reveal that (Y32)LCDR1, (R31)HCDR1, (W33)HCDR1, (R98)HCDR3, (L97)HCDR3, and (Y100)HCDR3 are responsible for approximately 80% of the binding free energy. Here we evaluate the role of these residues along with a group of basic residues (K62, K64, R24, K52) within the context of the binding mechanism. Binding of 11F8 takes place in two steps. In the first step, the overall positive charge of the antigen binding site attracts the negatively charged DNA to form an encounter complex that is stabilized by two salt bridges and a hydrogen bond. The second step is a slow process in which minor conformational changes occur. During this step, aromatic side chains become desolvated, presumably through stacking interactions involving two thymine bases within the DNA recognition epitope. Although the stability of the complex arises primarily from interactions formed in the second step, sequence specificity results from interactions with residues involved in both steps. These studies also show that the way in which 11F8 achieves high affinity sequence-specific binding is more closely related to RNA binding proteins than those that bind DNA and point to strategies for disrupting DNA binding that could prove to be therapeutically useful. 相似文献
6.
H Yokoyama R Mizutani Y Satow Y Komatsu E Ohtsuka O Nikaido 《Nucleic acids symposium series》1999,(42):267-268
Crystal structures of the 64M-2 antibody Fab fragment complexed with DNA photoproducts of dT(6-4)T and dTT(6-4)TT, and of the 64M-3 Fab fragment complexed with dT(6-4)T were determined. The 5'-thymine base of the bound dT(6-4)T ligand is in a half-chair conformation, and its base plane is nearly perpendicular to the planar 3'-pyrimidone base. The 64M-2 and 64M-3 Fabs have a common structure suitable for accommodating the dT(6-4)T ligand. In each of the antigen binding sites of the 64M-2 and 64M-3 Fabs, basic residues of His 35H and Arg 95H are located at the bottom of the binding pocket, and are hydrogen-bonded to the base moieties of dT(6-4)T. Two water molecules are involved in the interactions that intervene between the base moieties and the binding site. Aromatic residues of Trp 33H and Tyr 100iH form a side-wall of the pocket and are in van der Waals interactions with the base moieties. The Trp 33H side-chain is placed in parallel to the 3'-pyrimidone base, and the Tyr 100iH side-chain is nearly perpendicular to the 5'-thymine base. His 27dL, Tyr 32L, Leu 93L, and Ser 58H forming another side-wall are located in the vicinity of the sugar-phosphate backbone. In the 64M-2 Fab complex with dTT(6-4)TT, 5'- and 3'-side phosphate groups are also involved in interaction with Fab residues. 相似文献
7.
B Mao Z Gu A Gorin J Chen B E Hingerty S Amin S Broyde N E Geacintov D J Patel 《Biochemistry》1999,38(33):10831-10842
Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix. 相似文献
8.
The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft. 相似文献
9.
The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair. 相似文献
10.
B Parhami-Seren M Viswanathan R K Strong M N Margolies 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(9):5129-5135
Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding. 相似文献
11.
The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (DeltaG degrees ), enthalpy (DeltaH degrees ), entropy (DeltaS degrees ), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997) Nucleic Acids Res. 25, 1442-1449]. The contribution of separate residues of HCDR3 to ssDNA interaction was investigated. Analysis of the changes in DeltaH degrees and TDeltaS degrees, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases in TDeltaS degrees ( )()by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)(15). The observed linear correlation of Z and DeltaG degrees ( )()of nonelectrostatic interactions (DeltaG degrees (nonel)) at the anti-ssDNA combining site was used for the estimation of the specific DeltaG degrees (nonel) [-20 to -25 cal/(mol.A(2))], the average contact area (450-550 A(2)), the maximal Z (6-7), and the limit in affinity under standard cation concentrations [(0.5-1) x 10(8) M(-)(1)] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab-DNA interactions. 相似文献
12.
R Ivarie 《Nucleic acids research》1987,15(23):9975-9983
Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings. 相似文献
13.
Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibodydsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage. 相似文献
14.
Conformation and dynamics of the Pribnow box region of the self-complementary d(C-G-A-T-T-A-T-A-A-T-C-G) duplex in solution 总被引:1,自引:0,他引:1
Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called Pribnow 12-mer), which contains a TATAAT Pribnow box and a central core of eight dA X dT base pairs. The exchangeable imino and nonexchangeable base protons have been assigned from one-dimensional intra and inter base pair nuclear Overhauser effect (NOE) measurements. Premelting conformational changes are observed at all the dA X dT base pairs in the central octanucleotide core in the Pribnow 12-mer duplex with the duplex to strand transition occurring at 55 degrees C in 0.1 M phosphate solution. The magnitude of the NOE measurements between minor groove H-2 protons of adjacent adenosines demonstrates that the base pairs are propeller twisted with the same handedness as observed in the crystalline state. The thymidine imino proton hydrogen exchange at the dA X dT base pairs has been measured from saturation recovery measurements as a function of temperature. The exchange rates and activation barriers show small variations among the four different dA X dT base pairs in the Pribnow 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
16.
Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired. 相似文献
17.
Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA. 总被引:1,自引:1,他引:0
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Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs. 相似文献
18.
《Gene》1996,168(1):9-14
The display of antibody (Ab) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute the most to Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100%. A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR. 相似文献
19.
Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids 总被引:5,自引:0,他引:5
Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse. 相似文献
20.
S C Kao E V Bobst G T Pauly A M Bobst 《Journal of biomolecular structure & dynamics》1985,3(2):261-268
A set of differently spin labeled (dT)n is used to evaluate thymidine dynamics and some of the structural features in a (dT)n-gene 5 protein complex. ESR evidence is presented that only one of the four thymidine residues bound in the DNA binding channel shows strong immobilization, whereas the other three display significant mobility of the order of nanoseconds. It is hypothesized that the accessability of such mobile bases could be critical to the recognition of the (dT)n-gene 5 protein complex in auxiliary interactions with other proteins and competitive DNAs. 相似文献