Preparation, Characterization and Crystallization of an Antibody Fab Fragment that Recognizes RNA

Pokkuluri, P.R.; Bouthillier, François; Li, Yunge; Kuderová, Alena; Lee, Jeremy; Cygler, Mirosław

doi:10.1006/jmbi.1994.1654

Cited by 61 publications

(50 citation statements)

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“…The structure of 34E4 was determined by molecular replacement by using MERLOT (25) to rapidly screen Ϸ250 Fab structures for potential solutions to the rotation function. Among the 10 top solutions, the murine ribonucleotide-binding antibody Jel103 (26) was found to be the best model by using the program AMORE (27) (29). The hapten could easily be built into unambiguous difference electron density maps for both complex structures.…”

Section: Methodsmentioning

confidence: 99%

Structural origins of efficient proton abstraction from carbon by a catalytic antibody

Debler

Ito

Seebeck

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-Å resolution shows that a combination of hydrogen bonding, stacking, and van der Waals interactions is exploited to position both the base, Glu H50 , and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-Å structure of the Glu H50 Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp L91 and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acidbase pairs capable of bifunctional catalysis.crystal structure ͉ base catalysis ͉ proton transfer ͉ medium effects ͉ orientation effects P roton abstraction from carbon constitutes a fundamental process catalyzed by numerous enzymes, including isomerases, epimerases, racemases, lyases, and some synthases (1). Although heterolytic COH bond cleavage is a kinetically and thermodynamically demanding reaction, deprotonation catalysts rank among the most efficient enzymes known. For example, triosephosphate isomerase, ketosteroid isomerase, and fumarase operate at the diffusion limit (2). The mechanisms by which proteins that contain only weak acids and bases accelerate proton transfers by such enormous factors has long intrigued biochemists (3). Crystal structures and biochemical experiments of such enzymes have highlighted the importance of precisely positioned functional groups with optimized pK a s (3-5), but the precise origins of their high activities remain controversial (6).Valuable insight into the factors that contribute to efficient proton abstraction can be gained from the base-promoted Kemp elimination (1 3 3, Fig. 1), a widely used model system that is sensitive to base strength and solvent environment (7-9). This reaction is also susceptible to catalysis by antibodies (10, 11), albumins (12-14), polyethyleneimine ''synzymes'' (15, 16), organic hosts (17), cationic vesicles (18), and even natural coals (19). Antibody 34E4, which was raised against the cationic 2-aminobenzimidazolium derivative 4, is particularly effective in this regard (11). It promotes the decomposition of benzisoxazole 1 with Ͼ10 3 turnovers per active site and achieves a rate acceleration of 10 6 over backgrou...

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Section: Methodsmentioning

confidence: 99%

Structural origins of efficient proton abstraction from carbon by a catalytic antibody

Debler

Ito

Seebeck

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The importance of particular amino acids from both chains of BV0401 was confirmed by mutagenesis of bacterially expressed scFv (Rumbley et al, 1993). In an antibody induced by immunization with poly(I), the L chain differed only at residue 96 from the BV0401 L chain and contributed more contacts than the H chain to binding mononucleotides of hypoxanthine (Pokkuluri et al, 1994). The importance of the junctional L chain residue 96 in forming antigen-binding sites has been demonstrated for anti-hapten antibodies (Davies et al, 1990;Haseman and Capra, 1991) and some anti-DNA antibodies Polymenis and Stollar, 1995), even when the H chain alone can bind the antigen (Polymenis and Stollar, 1995).…”

Section: The Roles Of H and L Chain V Regionsmentioning

confidence: 99%

Heavy chain dominance in the binding of DNA by a lupus mouse monoclonal autoantibody

Jang

Lecerf

Stollar

1996

Molecular Immunology

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“…This dearth of information reflects our inability to elicit antibodies against RNA by using traditional approaches. RNA appears to lack immunogenic potency (18), and its susceptibility to nuclease degradation prohibits direct immunization of animals, which precludes the use of hybridoma technology for large structured RNAs. A robust platform for obtaining antibodies against RNA would enable the investigation of RNA biology by using approaches analogous to those that have proven to be extremely effective for the study and therapeutic manipulation of protein-protein interactions.…”

mentioning

confidence: 99%

Synthetic antibodies for specific recognition and crystallization of structured RNA

Tereshko

Frederiksen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

117

157

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Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigenbinding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementaritydetermining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.antigen-binding fragments ͉ x-ray crystallography A ntibodies are integral components of the immune system and represent a rapidly growing sector of the biotechnology industry (1, 2). Clinically, antibodies serve as diagnostic markers for disease antigens and play increasingly important roles as therapeutic agents for a wide range of diseases (3). Antibodies also provide invaluable biomedical research tools, serving to define the components and functions of macromolecular complexes, to establish cellular distributions of proteins, and to facilitate structural analysis as chaperones for crystallization of membrane proteins (4-6). Hybridoma and other technologies have yielded antibodies against a vast array of specific antigens (2). An enormous body of literature documents the molecular details of antibody interactions with a variety of antigens, including proteins (7), polysaccharides (8), and small haptens (9). However, much less information (and, in particular, no structural information) exists for antibody-RNA interactions.The relative absence of antibodies that bind RNA from the immunologic repository is striking, especially considering that recent genome-wide analyses of the metazoan transcriptome have revealed the presence of vast numbers of noncoding RNAs, including silencing RNAs, riboswitches, catalytic RNAs, and a multitude of other functional RNA moleucles (10,11). A large number of these RNAs adopt complex three-dimensional architectures that frequently act in complex with proteins to mediate their biological function (12, 13). Nevertheless, with the exception of a handful of examples, mostly isolated from the sera of autoimmune patients (14-17), we know little about anti-RNA antibodies and their recognition of nucleic acids. This dearth of information reflects our inability to elicit antibodies against RNA by using traditional approaches. RNA appears to lack immunogenic potency...

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Preparation, Characterization and Crystallization of an Antibody Fab Fragment that Recognizes RNA

Cited by 61 publications

References 0 publications

Structural origins of efficient proton abstraction from carbon by a catalytic antibody

Structural origins of efficient proton abstraction from carbon by a catalytic antibody

Heavy chain dominance in the binding of DNA by a lupus mouse monoclonal autoantibody

Synthetic antibodies for specific recognition and crystallization of structured RNA

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