No abstract

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.

Although affinity maturation constitutes an integral part of T-dependent humoral responses, its structural basis is less well understood. We compared the physicochemical properties of antigen binding of several independent antibody panels derived from both germline and secondary responses. We found that antibody maturation essentially reflects modulations in entropy-control of the association, but not dissociation, step of the binding. This influence stems from variations in conformational heterogeneity of the antigen-combining site, which in turn regulates both the affinity and specificity for antigen. Thus, the simple device of manipulating conformational flexibility of paratope provides a mechanism wherein the transition from a degenerate recognition capability to a high-fidelity effector response is readily achieved, with the minimum of somatic mutations.

Correlation between the promiscuity of the primary antibody response and conformational flexibility in a germline antibody was addressed by using germline antibody 36-65. Crystallographic analyses of the 36-65 Fab with three independent dodecapeptides provided mechanistic insights into the generation of antibody diversity. While four antigen-free Fab molecules provided a quantitative description of the conformational repertoire of the antibody CDRs, three Fab molecules bound to structurally diverse peptide epitopes exhibited a common paratope conformation. Each peptide revealed spatially different footprints within the antigen-combining site. However, a conformation-specific lock involving two shared residues, which were also associated with hapten binding, was discernible. Unlike the hapten, the peptides interacted with residues that undergo somatic mutations, suggesting a possible mechanism for excluding "nonspecific" antigens during affinity maturation. The observed multiple binding modes of diverse epitopes within a common paratope conformation of a germline antibody reveal a simple, yet elegant, mechanism for expanding the primary antibody repertoire.

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of “evolvability.”