Infant Abs induced by viruses exhibit poor functional activity compared with those of adults. The human B cell response to rotavirus is dominated by use of the VH1–46 gene segment in both adults and infants, but only adult sequences are highly mutated. We investigated in detail the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in rotavirus-specific human Abs encoded by the immunodominant VH1–46 gene segment. Adult Abs achieved enhanced binding through naturally occurring somatic mutations in the H chain CDR2 region that conferred a markedly prolonged off-rate and a desirable increase in antiviral potency. Three-dimensional cryoelectron microscopy studies of Ag-Ab complexes revealed the mechanism of viral inhibition to be the binding of high-affinity Abs at the viral RNA release pore in the double-layer particle. These structure-function studies suggest a molecular basis for the poor quality of Abs made in infancy following virus infection or immunization.

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E 1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with Ϸ40% and full E 1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E 1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E 1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E 2) that tethers E1 to the E2 core increases in length from Ϸ50 to 75 Å, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E 1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E 1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.A central feature of eukaryotic pyruvate dehydrogenase complexes (PDCs) is a 60-mer core with the morphology of a pentagonal dodecahedron (1-4). The dihydrolipoamide acetyltransferase (E 2 ) component serves as a scaffold to which the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ) components are attached. The E 2 dodecahedron consists of sets of three tightly bound subunits at each of its 20 vertices. The trimers are interconnected by 30 tenuous and flexible bridges that enable the core to breathe as evidenced by its size variability of Ϸ17% (240-280 Å in diameter) at room temperature (5).3D reconstructions of subcomplexes of the Saccharomyces cerevisiae E 2 core have revealed that 12 E 3 components are attached by a binding protein (BP) inside the 12 pentagonal openings of the cage-like E 2 (6), whereas the E 1 components form a shell that surrounds the underlying E 2 core (7-9). The reconstructions show that the 60 E 2 subunits are organized in 20 trimers. Each E 2 subunit contains an Ϸ50-Å-long linker that anchors an E 1 tetramer. Three of these linkers emanate from the outside edges of the triangular-shaped base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains of E 2 and the E 1 and E 2 active sites. We proposed that the lipoyl domain and its tether (swinging arm) rotates about the E 1 -binding domain of E 2 , which...

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