Specific Recognition of a DNA Immunogen by its Elicited Antibody

Sanguineti, Santiago; Crowley, Juan M. Centeno; Merlo, María F. Lodeiro; Cerutti, María Laura; Wilson, Ian A.; Goldbaum, Fernando A.; Stanfield, Robyn L.; Prat‐Gay, Gonzalo de

doi:10.1016/j.jmb.2007.04.046

Cited by 13 publications

(33 citation statements)

References 55 publications

(59 reference statements)

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“…As discussed by Sanguineti et al, this motif is utilized by ED-10 to bind a cytosine ring. 26 Thus, all Fab-DNA crystal structures determined to date feature this binding motif.…”

Section: Comparison To Other Fab-dna Crystal Structuresmentioning

confidence: 99%

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Impact of DNA Hairpin Folding Energetics on Antibody–ssDNA Association

Bottoms

Henzl

et al. 2007

Journal of Molecular Biology

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Deposition of anti-DNA antibodies in the kidney contributes to the pathogenesis of the autoimmune disease, systemic lupus erythematosus. Antibodies that bind to hairpin-forming DNA ligands may be particularly prone to deposition. Here we report the first structure of a Fab complexed with hairpin-forming DNA. The ligand used for co-crystallization is 5'-d [CTG(CCTT)CAG]-3', which has a predicted hairpin structure consisting of a four-nucleotide loop (CCTT) and a stem of three base-pairs. The 1.95 A resolution crystal structure of Fab DNA-1 complexed with this ligand shows that the conformation of the bound ligand differs radically from the predicted hairpin conformation. The three base-pairs in the stem are absent in the bound form. The protein binds to the last six nucleotides at the 3' end of the ligand. These nucleotides form a loop (TTCA) closed by a G:C base-pair in the bound state. Stacking of aromatic side-chains against DNA bases is the dominant interaction in the complex. Interactions with the DNA backbone are conspicuously absent. Thermodynamics of binding are examined using isothermal titration calorimetry. The apparent dissociation constant is 4 microM, and binding is enthalpically favorable and entropically unfavorable. Increasing the number of base-pairs in the DNA stem from three to six decreases binding affinity. These data suggest a conformational selection binding mechanism in which the Fab binds preferentially to the unstructured state of the ligand. In this interpretation, the ligand binding and ligand folding equilibria are coupled, with lower hairpin stability leading to greater effective binding affinity. Thus, pre-organization of the DNA loop into the preferred binding conformation does not play a major role in complexation. Rather, it is argued that the stem of the hairpin serves to reduce the degrees of freedom in the free DNA ligand, thereby limiting the entropic cost attendant to complexation with the Fab.

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“…As discussed by Sanguineti et al, this motif is utilized by ED-10 to bind a cytosine ring. 26 Thus, all Fab-DNA crystal structures determined to date feature this binding motif.…”

Section: Comparison To Other Fab-dna Crystal Structuresmentioning

confidence: 99%

“…26 The DNA-1/LIG5-14 structure is unique in that the bound ligand forms a closed loop featuring a base-pair, whereas the ligands in the other structures adopt linear conformations.…”

Section: Comparison To Other Fab-dna Crystal Structuresmentioning

confidence: 99%

Impact of DNA Hairpin Folding Energetics on Antibody–ssDNA Association

Bottoms

Henzl

et al. 2007

Journal of Molecular Biology

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“…Surprisingly, although there are at least 800 structures of Fab fragments in the Protein Data Bank, these include only a handful of structures for DNA or RNA binding Fab fragments. Fabs BV04‐01, DNA‐1, and ED‐10, are all available bound to small pieces of single stranded DNA and in the unliganded form. The structure of the isolated V L domain from the DNA hydrolyzing scFv 3D8 has also been reported .…”

Section: Resultsmentioning

confidence: 99%

Crystal structure determination of anti‐DNA Fab A52

Stanfield

Eilat

2014

Proteins

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A52 is a murine monoclonal antibody isolated from autoimmune New Zealand Black/New Zealand White F1 mice that recognizes single and double stranded DNA. This mouse strain spontaneously develops systemic lupus erythematosus-like symptoms and has served as a model for that disease for many years. The 1.62 Å crystal structure of the A52 Fab fragment reveals an H3 complementarity determining region with four closely spaced arginine residues, creating a positively charged surface to accommodate bound DNA.

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“…This was not surprising as S55-3 and S55-5 form a small cavity that is unlikely to accommodate an oligonucleotide, and furthermore, the binding sites lack the stacking interactions with nucleotide bases via Tyr and Trp residues observed in anti-nucleotide antibodies (48,(81)(82)(83).…”

Section: Antigenmentioning

confidence: 99%

The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups

Haji-Ghassemi

Müller‐Loennies

Rodrı́guez

et al. 2016

Journal of Biological Chemistry

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Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies.Bacterial Gram-positive and Gram-negative infections can lead to septic shock, with estimates as high as one million annual cases in the United States with a mortality rate as high as 50% (1-3). The amphipathic lipopolysaccharide (LPS) responsible for Gram-negative-induced septic shock is normally shed from the bacterial outer membrane but can be released in great amounts upon cell death (4). Lipid A, the endotoxic principle of LPS, is an acylated glucosamine phosphate disaccharide that anchors the LPS molecule to the bacterial outer membrane (5, 6). The presence of intact LPS (or lipid A) in blood can induce a potentially fatal inflammatory cascade in humans (7), initiated by the formation of a signaling complex of the lipid A with Toll-like receptor 4 (TLR4) and co-receptor myeloid differentiation factor 2 (MD-2) (8 -11).Efforts to develop therapeutic antibodies to inhibit the formation of the LPS⅐TLR4⅐MD-2 complex by sequestering LPS have proved challenging (12)(13)(14). Although antibodies specific for the various LPS components have been reported (15-24), the structural variation in the core and O-polysaccharide regions together with the rapid onset of septic shock have hindered their introduction into clinical use (4, 12, 25-27). To date, only the inner core binding mAb WN1 222-5 has been ...

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Specific Recognition of a DNA Immunogen by its Elicited Antibody

Cited by 13 publications

References 55 publications

Impact of DNA Hairpin Folding Energetics on Antibody–ssDNA Association

Impact of DNA Hairpin Folding Energetics on Antibody–ssDNA Association

Crystal structure determination of anti‐DNA Fab A52

The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups

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