Abstract
Antigen binding by antibodies requires precise orientation of the complementarity- determining region (CDR) loops in the variable domain to establish the correct contact surface. Members of the family Camelidae have a modified form of immunoglobulin gamma (IgG) with only heavy chains, called Heavy Chain only Antibodies (HCAb). Antigen binding in HCAbs is mediated by only three CDR loops from the single variable domain (VHH) at the N-terminus of each heavy chain. This feature of the VHH, along with their other important features, e.g., easy expression, small size, thermo-stability and hydrophilicity, made them promising candidates for therapeutics and diagnostics. Thus, to design better VHH domains, it is important to thoroughly understand their sequence and structure characteristics and relationship. In this study, sequence characteristics of VHH domains have been analysed in depth, along with their structural features using innovative approaches, namely a structural alphabet. An elaborate summary of various studies proposing structural models of VHH domains showed diversity in the algorithms used. Finally, a case study to elucidate the differences in structural models from single and multiple templates is presented. In this case study, along with the above-mentioned aspects of VHH, an exciting view of various factors in structure prediction of VHH, like template framework selection, is also discussed.
Highlights
Immunoglobulin Gamma (IgG) is a major component of the immune system in vertebrates
Our study focuses on VHH domains, since others have compared a more limited number of sequences (90) and always compare with VH as a priority (Mitchell & Colwell, 2018b)
Our paper in addition to reaffirming the sequences-structure characteristics of VHH domains reported in the recent literature, makes unique observations regarding (i) the variation in the amino acid signature in the FR2 region, (ii) conservation of β-strands and presence of other kinds of secondary structures, (iii) sheds light on conformations of di sulphide bridges and (iv) inter and intra cluster variations from PyIgclassify Complementary Determining Regions (CDR) clusters in terms of local conformations using Protein Blocks
Summary
Immunoglobulin Gamma (IgG) (see Fig. 1A) is a major component of the immune system in vertebrates. Members of the family Camelidae have a modified form of IgG, called Heavy Chain only Antibodies (HCAbs). HCAbs, as their name suggests, are completely devoid of (i) light chains and (ii) CH1 domain in the heavy chain (see Fig. 1B) (Hamers-Casterman et al, 1993). The N-terminal domain of each chain of HCAb, named VHH, is functional when expressed independently. A VHH domain is 20 times smaller than complete IgG, ranging from 120 to 150 amino acids in length. 1C–1E) has only 3 Complementary Determining Regions (CDR) to bind to their antigens. These loops connect the more structured regions called the Framework Regions (FR), considered sequentially and structurally well conserved
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