Structural Diversity of Ultralong CDRH3s in Seven Bovine Antibody Heavy Chains

Abstract

Antigen recognition by mammalian antibodies represents the most diverse setting for protein-protein interactions, because antibody variable regions contain exceptionally diverse variable gene repertoires of DNA sequences containing combinatorial, non-templated junctional mutational diversity. Some animals use additional strategies to achieve structural complexity in the antibody combining site, and one of the most interesting of these is the formation of ultralong heavy chain complementarity determining region 3 loops in cattle. Repertoire sequencing studies of bovine antibody heavy chain variable sequences revealed that bovine antibodies can contain heavy chain complementarity determining region 3 (CDRH3) loops with 60 or more amino acids, with complex structures stabilized by multiple disulfide bonds. It is clear that bovine antibodies can achieve long, peculiarly structured CDR3s, but the range of diversity and complexity of those structures is poorly understood. We determined the atomic resolution structure of seven ultralong bovine CDRH3 loops. The studies, combined with five previous structures, reveal a large diversity of cysteine pairing variations, and highly diverse globular domains.

Formation of ultralong DH regions through genomic rearrangement

“…The homologous DH regions are cysteine rich, and diversity can be generated through both germline and somatically generated cysteines, which can form a diverse array of potential disulfide bonded loops[23, 35, 36]. In the knob region of ultralong CDR H3s, a diversity of disulfide bond patterns has been observed in several crystal structures [23, 25, 26], and mutations to and from cysteine have been confirmed through deep sequence analysis. Thus, novel cysteines encoded in the DH regions contributes to structural diversity in bovine antibodies.…”

“…Bovines are remarkable in having very long CDR H3 regions[14–24], with an average length of 26 amino acids [16] but with an exceptionally long subset of the repertoire (the “ultralong” CDR H3 antibodies) that can have CDR H3 lengths of up to seventy amino acids. These CDR H3 regions form their own independently folding mini domains comprised of a β-ribbon “stalk” that protrudes far from the typicalparatope surface upon which sits a disulfide-bonded “knob”[21, 23, 25, 26]. Cows are the only species thus far investigated that can produce a broadly neutralizing antibody response against HIV, which is characterized by ultralong CDR H3 regions that penetrate the glycan shield of the spike protein to bind a conserved broadly neutralizing epitope in the CD4 binding region [27].…”

Broadly Neutralizing Bovine Antibodies: Highly Effective New Tools against Evasive Pathogens?

“…However, due to its presence in the region encoding the stem and its propensity for encoding hydrophilic residues [18], this non-germline sequence can likely alter the orientation of the knob domain while maintaining the β−strand structure. This feature may therefore change the local flexibility and geometry without compromising the overall stem stability [46]. Seven recent structures reveal that small changes (<7 • ) in the angle of the stalk can lead to large changes in the orientation of the putative antigen binding site [46].…”

“…This feature may therefore change the local flexibility and geometry without compromising the overall stem stability [46]. Seven recent structures reveal that small changes (<7 • ) in the angle of the stalk can lead to large changes in the orientation of the putative antigen binding site [46]. Notably, a threonine is nearly always found at the second position of this junctional insertion and is positioned directly opposite alternating tyrosines (YxYxY) encoded in the germline and present in the descending side of the stem in the 3D structure [18,47].…”

“…The ultralong CDR H3 structure was first characterised in detail in two cryo-EM structures [18] and has since been further analysed by Stanfield et al [47], Dong et al [46] and reviewed by others [19,48]. Briefly, the stalk of bovine long CDR H3 Abs consists of anti-parallel βstrands that protrude away from the main Ab structure, akin to the strands that stabilize the anionic protrusions of some human BNAbs.…”