Conformational isomerism and the diversity of antibodies.

Abstract

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...

Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

“…However, recent crystallographic and thermodynamic analyses of both germline and mature Ab-Ag complexes have suggested that the Ab repertoire can be further enhanced at the tertiary structural level (7,14,18,19,34,35). The present study provides an elegant correlation of CDR conformational versatility in a germline Ab with its multispecificity.…”

“…Our data illustrate the importance of paratope adaptability *Structural Biology Unit, National Institute of Immunology, New Delhi 110 067, India; and † Regional Centre for Biotechnology, Gurgaon 122 016, India for facilitating polyreactivity of germline mAb through systematically designed crystallographic experiments. The present analysis, in light of earlier studies (1,5,8,9,(14)(15)(16)(17)(18)(19), suggests the possibility that generation of diversity in the primary Ab repertoire may involve multiple mechanisms in a hierarchical order. The proposed model may account for enhanced diversity of primary immune receptors without increasing the size of the germline gene repertoire.…”

“…Six structures of the germline mAb BBE6.12H3, in different chemical environments, quantitatively define the conformational repertoire of the CDRs, providing a rich data set for examining paratope topologies, although thermodynamic and computational studies have provided interesting clues regarding the generation of diversity in primary Ab response (5,19,33,34). Similarly, crystallographic and kinetic analyses of mature Abs SPE7 and NQ22/61.1, respectively, have also suggested conformational isomerism (7,19). Our data consisting of six BBE6.12H3 Fab structures-two Ag free and four Ag bound-provided a rich source of information reflecting correlation of paratope conformational flexibility and polyreactivity.…”

Physicochemical bases for protein folding, dynamics, and protein-ligand binding

“…Furthermore, these states/substates can interconvert, with the transition rate determining the probability distribution and population size of these states. Based on the thermodynamic and kinetic properties of proteins, a more "real" model, the conformational selection model [87][88][89] was proposed to explain the protein-ligand binding mechanism at the molecular population level.…”

Binding of Phenylphosphocholine−Carrier Conjugates to the Combining Site of Antibodies Maintains a Conformation of the Hapten

“…This loss in flexibility is due to an increased effective correlation time (in this case, probable loss of flexibility of the binding site for hapten), leading to a larger negative NOE (that is, the cross-peaks are in the same phase as the diagonal peaks and have an increased intensity). Conformational mobility of the antibody combining site has been proposed as a possible mechanism for cross-reactivity and multiphasic binding kinetics (Foote & Milstein, 1994). Such a mechanism may be common.…”