Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation

Vuk-Pavlović, S.; Blatt, Yoav; Glaudemans, Cornelis P.J.; Lancet, Doron; Pecht, Israel

doi:10.1016/s0006-3495(78)85353-3

Cited by 24 publications

(23 citation statements)

References 12 publications

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“…Bi-or triphasic binding kinetics was first described for anti-hapten antibodies (35,37,41,42), but have not previously been described for antibody-protein associations. The observation that anti-DNP myeloma proteins exhibit biphasic or triphasic kinetics is particularly relevant, as the antibody MOPC460 expresses V H 36-60 and Vκ23 genes and thus is structurally related to the anti-HEL antibodies described here (43,44).…”

Section: Evidence For a Two-mentioning

confidence: 99%

Mutations of an Epitope Hot-Spot Residue Alter Rate Limiting Steps of Antigen−Antibody Protein−Protein Associations

Lipschultz

Srinivasan

et al. 2001

Biochemistry

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The antibodies, HyHEL-10 and HyHEL-26 (H10 and H26, respectively), share over 90% sequence homology and recognize with high affinity the same epitope on hen egg white lysozyme (HEL) but differ in degree of cross-reactivity with mutant lysozymes. The binding kinetics, as measured by BIAcore surface plasmon resonance, of monovalent Fab from both Abs (Fab10 and Fab26) to HEL and mutant lysozymes are best described by a two-step association model consistent with an encounter followed by docking that may include conformational changes. In their complexes with HEL, both Abs make the transition to the docked phase rapidly. For H10, the encounter step is rate limiting, whereas docking is also partially rate limiting for H26. The forward rate constants of H10 are higher than those of H26. The docking equilibrium as well as the overall equilibrium constant are also higher for H10 than for H26. Most of the free energy change of association (Delta G degrees) occurs during the encounter phase (Delta G1) of both Abs. H10 derives a greater amount and proportion of free energy change from the docking phase (Delta G2) than does H26. In the H10--HEL(R21Q) complex, a significant slowing of docking results in lowered affinity, a loss of most of Delta G2, and apparently faster dissociation. Slower encounter and docking cause lowered affinity and a loss of free energy change primarily in the encounter step (Delta G1) of H26 with mutant HEL(R21Q). Overall, in the process of complex formation with lysozyme, the mutations HEL(R21X) affect primarily the docking phase of H10 association and both phases of H26. Our results are consistent with the interpretation that the free energy barriers to conformational rearrangement are highest in H26, especially with mutant antigen.

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Section: Evidence For a Two-mentioning

confidence: 99%

Mutations of an Epitope Hot-Spot Residue Alter Rate Limiting Steps of Antigen−Antibody Protein−Protein Associations

Lipschultz

Srinivasan

et al. 2001

Biochemistry

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“…To our knowledge, no triphasic antibody-hapten reaction has previously been reported. The absence of a third phase in earlier studies (10)(11)(12)(13)(14) has been attributed variously to experimental difficulties in detecting very short relaxation times or low amplitudes, or to the system approximating a limiting case of Mechanism I. The operation of Mechanism I in these cases was inferred from quantitative incongruities in other parameters, such as independently measured equilibrium constants or relaxation amplitudes.…”

mentioning

confidence: 99%

“…Kinetic analysis avoids this particular ambiguity. Indeed, studies on several myeloma proteins have demonstrated kinetically distinct conformational isomers (10)(11)(12)(13). It could be argued that myeloma cells have an uncertain ontogeny and are not subject to the immune maturation mechanisms encountered in a bona fide antigenic response; however, similar kinetic behavior was reported for an anti-fluorescein hybridoma (14).…”

mentioning

confidence: 99%

Conformational isomerism and the diversity of antibodies.

Foote

Milstein

1994

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

244

215

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The fact that one cell encodes a single antibody sequence does not ne arily mean that the resulting antibody folds into a single structure, although this is a common assumpton. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site.The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex haptenbinding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that lignd binding involved isomerization, as well as assoiative steps. The existence of an equilibrium between at least two antibody conformations, With ligands binding preferentially to one form, was deduced from the variation with hapten concentration ofthe apparent rate of each phase.Since Clonal Selection replaced the Instructive Theory as a working model of immunity, the axiom of one-lymphocyteone-antibody has been equated to one combining site (1-4). Experiments defining the basis of humoral immune diversity have emphasized combinatorial and somatic mutational processes that lead to covalent diversity of the antibody repertoire (5). Nevertheless, two independent lines of evidence exist for diversity at the level of antibody tertiary structure. Certain crystal structures of antibodies with and without ligands have shown conformation dimorphism, which could only partly be attributed to crystallization artifacts (6-8). Despite the rich detail of these structures, crystallographically detected conformational differences due to ligation are mechanistically ambiguous (9). Differences could arise from direct interaction with the ligand (induced fit) or, with equal plausibility, by preferential ligand binding to a preexisting subpopulation of antibody isomers. This distinction is of considerable immunological consequence, since two isomers in spontaneous equilibrium would both form part of the humoral repertoire, whereas an induced conformation existing only in an immune complex would not contribute to diversity in the same way. Kinetic analysis avoids this particular ambiguity. Indeed, studies on several myeloma proteins have demonstrated kinetically distinct conformational isomers (10-13). It could be argued that myeloma cells have an uncertain ontogeny and are not subject to the immune maturation mechanisms encountered in a bona fide antigenic response; however, similar kinetic behavior was reported for an anti-fluorescein hybridoma (14). Some of these cases demonstrate a preexisting isomeric equilibrium and others are consistent with an induced-fit mechanism. Notwithstanding this evidence that conformational polymorphism can occur, the isolated examples uncovered through either structural or kinetic approaches leave an uncertainty whether antibody isomerism is an oddity or a general and important factor in an immune response.Here we address the lack of a conte...

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“…In some studies (16,17), no conformational changes were observed upon binding of a small ligand. In others, conformational alterations correlated with hapten or antigen binding have been inferred on the basis of increased conformational stability ofthe Fab (18,19), decreased solubility (20), changes in the accessibility of bound hydrogens for solvent exchange (21), and results of numerous spectroscopic studies (22)(23)(24)(25)(26). In crystallographically based studies of conformational changes in the light-chain dimer Mcg, it was concluded that ligand binding caused alterations in both VL-VL and CL-CL interactions (27,28).…”

mentioning

confidence: 99%

Dual conformations of an immunoglobulin light-chain dimer: heterogeneity of antigen specificity and idiotope profile may result from multiple variable-domain interaction mechanisms.

Stevens

Chang

Schiffer

1988

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

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The structure of an immunoglobulin antigenbinding fragment (Fab) has been thought to be invariantly defined by well-coiserved amino acid residues in the variable domains of the heavy and light chains. These conserved residues enable folding of the polypeptide segments into the characteristic immunoglobulin fold domains and are the major controllers of interactions between domains. However, crystallographic studies of some immunoglobulin light-chain dimers have suggested and the crystallographic structure of the Fab in an Fab-neuraminidase complex may have proven that antibodies are not restricted to a single, invariant relative positioning of the two variable domains. We propose that in some cases the detailed quaternary structural relationships between the variable domains of heavy and light chains are not restricted to those of the canonical Fab. It is unclear whether alterations of these relationships occur only after complex formation with antigen or, if in ligand-free solution, Fab conformers might coexist in relative concentrations determined by isomerization rates. In the latter case, antibody-presenting lymphocytes may be polyspecific, and the specificity of lymphocytes might be modulated by anti-idiotopic antibodies complexed to cell surface receptors. In either case, the idiotopic repertoire displayed by an antibody or lymphocyte surface receptor might be changed by the presence or absence of antigen.Crystallographic studies of antigen-binding fragments (Fabs) have indicated that the principal determinants of quaternary structure are highly conserved domain-domain interactions (1)(2)(3)(4)(5) and that functional diversity originates from residue alternations, insertions, and deletions in segments of the heavy (H)-and light (L)-chain variable (V) domains designated hypervariable or complementarity determining regions (6). Several analogous studies with light-chain dimers have also supported the monomorphic conformation concept (7-10). However, in two instances (11,12), the observed Bence Jones protein conformations were anomalous; V-V domain interactions were found that had not been observed in Fabs. Although the relationships between these V domains were of significant interest in terms of the physical chemistry of antibody domain interactions, the physiological implications of the structures of the light-chain dimers formed by proteins Rhe (11)

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Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation

Cited by 24 publications

References 12 publications

Mutations of an Epitope Hot-Spot Residue Alter Rate Limiting Steps of Antigen−Antibody Protein−Protein Associations

Mutations of an Epitope Hot-Spot Residue Alter Rate Limiting Steps of Antigen−Antibody Protein−Protein Associations

Conformational isomerism and the diversity of antibodies.

Dual conformations of an immunoglobulin light-chain dimer: heterogeneity of antigen specificity and idiotope profile may result from multiple variable-domain interaction mechanisms.

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