Structure of digoxin

Go, K.; Kartha, G.; Chen, J. P.

doi:10.1107/s0567740880007297

Cited by 39 publications

(29 citation statements)

References 8 publications

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“…Although such rearrangements have been noted in other antibodies (Rini et al, 1992), comparison of the X-ray crystal structures of 26-10 Fab and the 26-10 Fab-digoxin complex reveals no significant differences in antibody structure upon binding (Jeffrey et al, 1993). The only observed difference between the crystal structure of digoxin (Go et al, 1980) and the structure of digoxin complexed with 26-10 (Jeffrey et al, 1993) is a rotation of the lactone ring of approximately 180" about the C17-C20 bond. Both of these lactone orientations have been observed in other cardiac glycoside crystal structures ( Go et al, 1980), but the high degree of complementarity between antibody and lactone allows only a single lactone conformation when complexed (Jeffrey et al, 1993).…”

Section: Molecular Modeling and Dockingmentioning

confidence: 73%

“…The only observed difference between the crystal structure of digoxin (Go et al, 1980) and the structure of digoxin complexed with 26-10 (Jeffrey et al, 1993) is a rotation of the lactone ring of approximately 180" about the C17-C20 bond. Both of these lactone orientations have been observed in other cardiac glycoside crystal structures ( Go et al, 1980), but the high degree of complementarity between antibody and lactone allows only a single lactone conformation when complexed (Jeffrey et al, 1993). We assume digoxin will complex with the mutants in the same orientation as it does with 26-10.…”

Section: Molecular Modeling and Dockingmentioning

confidence: 99%

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Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody

et al. 1994

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Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescenceactivated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone.The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc NUN Acud Sci USA 90: 10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

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Section: Molecular Modeling and Dockingmentioning

confidence: 73%

Section: Molecular Modeling and Dockingmentioning

confidence: 99%

Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody

et al. 1994

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“…Digoxin is a cardenolide-type steroid with an a,4-unsaturated lactone ring attached at C-17 and three P(1 -+ 4)-D-glycoside-linked digitoxoses attached at 0-3. It is a relatively large and rigid hapten, whose internal degrees of freedom are limited to rotations about the lactone-cardenolide bond (C-17 to C-20; Structure I) and about the digitoxose linkages (2). Many digoxin congeners of known chemical structure are available, including several with high-resolution crystal structures (2).…”

mentioning

confidence: 99%

26-10 Fab-digoxin complex: affinity and specificity due to surface complementarity.

Jeffrey

Strong

Sieker

et al. 1993

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

119

106

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We have determined the three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody 26-10 in the uncomplexed state at 2.7 A resolution and as a complex with digoxin at 2.5 A resolution.Neither the antibody nor digoxin undergoes any significant conformational changes upon forming the complex. Digoxin interacts primarily with the antibody heavy chain and is oriented such that the carbohydrate groups are exposed to solvent and the lactone ring is buried in a deep pocket at the bottom of the combining site. Despite extensive interactions between antibody and antigen, no hydrogen bonds or salt links are formed between 26-10 and digoxin. Thus the 26-10-digoxin complex is unique among the known three-dimensional structures ofantibody-antigen complexes in that specificity and high affinity arise primarily from shape complementarity.Digoxin (digoxigenin tridigitoxoside; Structure I) inhibits the Na+,K+-ATPase and is used in treatment of congestive heart failure (1). Digoxin is a cardenolide-type steroid with an a,4-unsaturated lactone ring attached at C-17 and three P(1 -+ 4)-D-glycoside-linked digitoxoses attached at 0-3. It is a relatively large and rigid hapten, whose internal degrees of freedom are limited to rotations about the lactone-cardenolide bond (C-17 to C-20; Structure Anti-digoxin antibodies have been used to measure serum digoxin levels as a guide to therapy and to treat otherwise fatal digoxin intoxication (3). Anti-digoxin antibodies complexed with structurally distinct digoxin congeners are also an excellent model system to study the structural basis of antibody specificity.The antibody 26-10 (IgG2a, K light chain) is a murine monoclonal antibody that exhibits high affinity (1010 M-1) for digoxin and varying specificity for digoxin congeners (4-6). Both the intact 26-10 antibody and the 26-10 Fab (antigenbinding fragment) reverse lethal digoxin toxicity in guinea pigs (7). Spontaneous and site-directed mutants of26-10 have been generated (6,8,9). The specificities of 26-10 and its mutants have been characterized for a wide range of digoxin congeners (6, 8, 9).We report the three-dimensional structures of the 26-10 Fab fragment at 2.7 A resolution and of the Fab-digoxin complex at 2.5 A resolution.i ¶ MATERIALS AND METHODS Antibody 26-10 was obtained from A/J mice immunized with a digoxin-human serum albumin conjugate (4). Purified antibody was cleaved by papain, and the Fab was purified by using ion-exchange chromatography (10). Crystals of the 26-10 Fab-digoxin complex were grown in the presence of a 3-fold molar excess of digoxin from 16% to 18% (wt/wt) polyethylene glycol (average Mr = 8000) with 1.7% (vol/vol) 2-methyl-2,4-pentanediol in 10 mM sodium phosphate at pH 7.5 (11

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“…1) is a useful hapten for studies of antibody complementarity as it approximates in size the antibody combining site, is uncharged, contains a rigid steroid nucleus, and has a known three-dimensional structure (16); multiple congeners of defined structure are known (17,18). Anti-digoxin antibodies are characterized by high affinity and varying specificity for related cardiac glycosides.…”

mentioning

confidence: 99%

“…This altered recognition of the steroid C ring 12-hydroxyl group among the three antibodies is also reflected in their relative binding to gitoxin (Table 1). Gitoxin, like digitoxin, lacks the 12-hydroxyl group but has an additional hydroxyl at position 16 To determine the structural changes responsible for antigen binding differences among the three hybridoma proteins, the complete V region sequences were determined by nucle- Proc. Natl.…”

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confidence: 99%

Variable region framework differences result in decreased or increased affinity of variant anti-digoxin antibodies.

Panka

Mudgett-Hunter

Parks

et al. 1988

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

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Rare spontaneous variants of the anti- Digoxin (digoxigenin tridigitoxose, Fig. 1) is a useful hapten for studies of antibody complementarity as it approximates in size the antibody combining site, is uncharged, contains a rigid steroid nucleus, and has a known three-dimensional structure (16); multiple congeners of defined structure are known (17,18). Anti-digoxin antibodies are characterized by high affinity and varying specificity for related cardiac glycosides. The anti-digoxin antibody 40-150 (18) was chosen for isolation of variants because of its sensitivity to the presence of a hydroxyl group at the 12 position on the steroid C ring of digoxin (Fig. 1) as it binds to digoxin more avidly than digitoxin, which lacks the hydroxyl group. We report the isolation and characterization from cell culture of a spontaneous antibody variant with reduced affinity due to a point mutation. A second-order variant demonstrated partial regain of binding due to a "compensatory" deletion of two amino acids from the framework region. MATERIAL AND METHODSCell Lines and Antibody Purification. The production of the murine A/J anti-digoxin hybridoma cell line (IgGl, K) has been reported (18). Production of 40-150 and variant hybridoma proteins in ascites was performed as described (17). Antibodies were purified from ascites or

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Structure of digoxin

Cited by 39 publications

References 8 publications

Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody

Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody

26-10 Fab-digoxin complex: affinity and specificity due to surface complementarity.

Variable region framework differences result in decreased or increased affinity of variant anti-digoxin antibodies.

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