A Comparative Analysis of the Immunological Evolution of Antibody 28B4

Abstract

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K d ) 25 µM) and 28B4 Fab (K d ) 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95 H Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. ( ) Science 279, 1929. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.The evolution of binding affinity in the immune system involves combinatorial and mutational processes, which in many aspects parallel those that occur in the natural evolution of enzymes. Consequently, the characterization of the affinity mat...

Structural evidence for substrate strain in antibody catalysis

“…4). This ''lock-and-key'' binding mode (22) of the affinity-matured antibody versus ''induced-fit'' binding mechanism (23) in the germ-line antibody was also observed in the antibodies 48G7, AZ28, and 28B4 (19,24,25) and further supports the hypothesis that the structural plasticity of germ-line antibodies is a critical factor in the ability of the immune system to evolve antibodies that bind many distinct chemical structures.…”

“…The binding properties (K d and k off ) of 7G12 Fab, germ-line Fab, and all of the mutants with NMP were measured by surface plasmon resonance on a Biacore 3000 biosensor following the published methods (19). BSA was conjugated with NMP and immobilized on a research grade CM5 sensor chip (Biacore).…”

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.…”

“…In the case of germline mAbs 28B4, 48G7, and AZ28, conformational rearrangement of CDRH3 has been shown to facilitate hapten binding. However, none of these studies have associated conformational flexibility of CDRs with degenerate specificity (14,(35)(36)(37).…”

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics

“…The dissociation constant K D , the dissociation rate constant k off , and the association rate constant k on of the Ab-Fl complexes were determined by using surface plasmon resonance on a Biacore 3000 biosensor (Biacore, Uppsala, Sweden) following published methods (38). Briefly, BSA was conjugated with Fl and immobilized on a research-grade CM5 sensor chip.…”