Reshaping Antibody Diversity

SummarySome species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β strand “stalk” that supports a structurally diverse, disulfide-bonded “knob” domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides

Functional analysis of the ubiquitin ligase Hrd1p with the ubiquitin-conjugating enzyme Ubc7p

Ubiquitin is a covalent protein tag that alters the stability or behavior of a growing list of proteins. Covalent attachment of ubiquitin to target proteins occurs through a cascade of enzymes: Ubiquitin is charged by a ubiquitin-activating enzyme (E1), and transferred to a ubiquitin-conjugating enzyme (E2). Then, transfer of ubiquitin from E2 to a target protein is brokered by a ubiquitin ligase (E3). A critical aspect of E3 function is the selection of a particular E2 to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function. During these studies we observed enhanced Ubc7p-dependent ubiquitination in the presence of soluble Cue1p, which interacts with Ubc7p in vivo. Soluble Cue1p promoted the transfer of ubiquitin from Ubc7p to other ubiquitin molecules in solution. We also observed that this stimulation of Ubc7p by Cue1p and the anchoring of Ubc7p to the ER membrane by Cue1p were both necessary for Ubc7p function in vivo. Ubc7p activation by Cue1p was observed at the ER and with Ubc7p relocated to a cytosolic E3. In total, these studies have substantially improved and expanded our understanding of how Hrd1p functions to degrade proteins in the E

Functional analysis of the ubiquitin ligase Hrd1p with the ubiquitin-conjugating enzyme Ubc7p

Ubiquitin is a covalent protein tag that alters the stability or behavior of a growing list of proteins. Covalent attachment of ubiquitin to target proteins occurs through a cascade of enzymes: Ubiquitin is charged by a ubiquitin-activating enzyme (E1), and transferred to a ubiquitin-conjugating enzyme (E2). Then, transfer of ubiquitin from E2 to a target protein is brokered by a ubiquitin ligase (E3). A critical aspect of E3 function is the selection of a particular E2 to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function. During these studies we observed enhanced Ubc7p-dependent ubiquitination in the presence of soluble Cue1p, which interacts with Ubc7p in vivo. Soluble Cue1p promoted the transfer of ubiquitin from Ubc7p to other ubiquitin molecules in solution. We also observed that this stimulation of Ubc7p by Cue1p and the anchoring of Ubc7p to the ER membrane by Cue1p were both necessary for Ubc7p function in vivo. Ubc7p activation by Cue1p was observed at the ER and with Ubc7p relocated to a cytosolic E3. In total, these studies have substantially improved and expanded our understanding of how Hrd1p functions to degrade proteins in the E