Molecular evolution of affinity and flexibility in the immune system

Abstract

The immune system responds to the introduction of foreign antigens by rapidly evolving antibodies with increasing affinity for the antigen (i.e., maturation). To investigate the factors that control this process at the molecular level, we have assessed the changes in flexibility that accompany ligand binding at four stages of maturation in the 4-4-20 antibody. Our studies, based on molecular dynamics, indicate that increased affinity for the target ligand is associated with a decreased entropic cost to binding. The entropy of binding is unfavorable, opposing favorable enthalpic contributions that arise during complex formation. Computed binding free energies for the various antibody-ligand complexes qualitatively reproduce the trends observed in the experimentally derived values, although the absolute magnitude of free-energy differences is overestimated. Our results support the existence of a correlation between high-affinity interactions and decreased protein flexibility in this series of antibody molecules. This observation is likely to be a general feature of molecular association processes and key to the molecular evolution of antibody responses. molecular recognition ͉ antibody ͉ ligand association ͉ binding ͉ entropy M olecular recognition plays a central role in many biological processes, including the regulation of development, cell signaling, and neutralization of foreign molecules by the immune system. Consequently, there is significant interest in elucidating the mechanisms involved in recognition processes from both experimental (1, 2) and theoretical (3, 4) perspectives; this subject is discussed in a number of reviews (5-7). Secreted antibodies (Abs) play a central role in the immune response and bind to their target antigens with both high affinity and specificity. These molecules serve as excellent models with which to examine the mechanisms of molecular recognition. The Ab 4-4-20 is particularly useful in this regard, as it binds to the chromophore fluorescein (FLU) with high affinity (8). Interaction of 4-4-20 with FLU has greatly facilitated structural, kinetic and, thermodynamic characterization of this Ab (9-12). Romesberg and colleagues (13-15) have characterized evolution of the immunological response for the well studied 4-4-20. They have deduced a sequence of mutations that engender increased affinity for the target ligand during the maturation process; evidence has been presented that the flexibility of the binding pocket decreases in concert with gains in affinity.As maturation proceeds, affinity for FLU increases going from the germ line (GL) Ab through two intermediates (IMs) (IM1 and IM2, respectively) until finally the highest affinity mature (AM) 4-4-20 Ab is attained. As determined by surface plasmon resonance, the dissociation constant of AM for FLU is 220 nM (14). IM2 has a dissociation constant of 400 nM and differs from AM in that residue 46 in the light chain is leucine (L L 46) rather than the valine (V L 46) present in the crystal structure. V L 46 does not interact with ...

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

“…The observed conformational variations in germline Ab-combining sites are consistent with the predictions based on thermodynamic studies and molecular dynamics simulations (5,33). On the basis of energy considerations, the conformations adopted by CDRH3 cannot represent a continuum of conformational space but, rather, correspond to a finite number of discrete states.…”

“…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).…”

Entropic contributions and the influence of the hydrophobic environment in promiscuous protein–protein association

“…The results of the current study, in which both the vibrational and conformational terms were measured, indicate that the approximation is not necessarily valid because the vibrational term was found to be the more dominant factor. Similar results have been reported by recent studies that suggested a narrowing of the energy landscape or the limitation of the flexibility of side-chain conformations contributes significantly to decrease in entropy upon association (17,20).…”

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

“…During antibody affinity maturation through somatic mutation in vivo, this flexibility is thought to be reduced leading to increased affinity and specificity. [4][5][6] With this in mind, one possibility is that conformational flexibility may have been introduced upon grafting the LD1 CDRs into the new human framework such that new interactions with C3d were enabled. Thus, rather than altering a direct interaction with C3 through affinity maturation, the changes made to CDR-H2 may have restricted the flexibility of this loop in a way that limited its interaction to FGFR4.…”