Analysis of nanobody paratopes reveals greater diversity than classical antibodies
Open accessAbstract
Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb-antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural features of the paratopes of both data sets. Our analysis finds that the set of Nb structures displays much greater paratope diversity, in terms of the structural segments involved in the paratope, the residues used at these positions to contact the antigen and furthermore the type of contacts made with the antigen. Our findings suggest a different relationship between contact propensity and sequence variability from that observed for Ab VH domains. The distinction between sequence positions that control interaction specificity and those that form the domain scaffold is much less clear-cut for Nbs, and furthermore H3 loop positions play a much more dominant role in determining interaction specificity.
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High-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of CDR3 loops (3-28 amino acids) compared to VH domains.
“Moreover, the high-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of the CDR3 loops (3–28 amino acids) compared to that of the VH domains [13].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [13]
The Introduction says VHH nanobodies achieve high binding affinities and have H3 loops around 15% longer than classical antibody H3 loops; the Conclusion links those loops to antigen contacts and interaction specificity. The paper does not establish that greater loop length supports high affinity, and the cited passages do not address V-NAR domains, a majority of loops, or the 3–28-amino-acid range (page numbers unavailable). (LLM, conf=0.85)
Experimental — accuracy not yet measuredRecent computational analyses of large sets of antibody and nanobody sequences and structures demonstrate that non-trivial systematic differences between these molecules exist.
“Recent computational analyses of large sets of antibody and nanobody sequences and structures demonstrate that non-trivial systematic differences between these molecules exist [231, 232].”
From Computational approaches to therapeutic antibody design: established methods and emerging trends (2020) · cites this paper as [231]
Analysis of large sets of nanobody-antigen co-crystal structures reveals that nanobody paratopes are made up of a much greater diversity of structural subunits, further increasing the modelling challenge.
“In addition, analysis of large sets of nanobody–antigen co-crystal structures reveals that nanobody paratopes are made up of a much greater diversity of structural subunits, further increasing the modelling challenge [231].”
From Computational approaches to therapeutic antibody design: established methods and emerging trends (2020) · cites this paper as [231]
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Computational approaches to therapeutic antibody design: established methods and emerging trends
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“these molecules is demonstrated by recent nanobody-specific databases and analyses [57, 58, 231, 232].”
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“Moreover, the high-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of the CDR3 loops (3–28 amino acids) compared to that of the VH domains [13].”
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“…diffraction structures and performed a survey of antibody-antigen contacts and contrasted them to protein-protein interfaces, which has been attempted several times before albeit with smaller datasets (Krawczyk et al. 2013; Kunik and Ofran 2013; Mitchell and Colwell 2018b, 2018a; Reis et al. 2022).”
- Authors
- Mitchell, L. S., & Colwell, L. J.
- Affiliations
- University of Cambridge (GB)
- Corresponding author
- Mitchell, Laura S
- Venue
- Protein Engineering Design and Selection
- Publisher
- Oxford University Press (OUP)
- Published
- 2018-07-01
- Volume
- 31
- Issue
- 7-8
- Pages
- 267-275
- ISSN
- 0269-2139 (ISSN-L)
- 1741-0126
- 1741-0134
- PMC
- PMC6277174
- OpenAlex
- W2883609523
- Cited by
- 138
- References
- 40
- Citations by year
- 2026: 17 · 2025: 20 · 2024: 23 · 2023: 22 · 2022: 20 · 2021: 15 · 2020: 7 · 2019: 12 · 2018: 2
- FWCI
- 6.22
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Protein Engineering Design and Selection (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Apollo (University of Cambridge) (submittedVersion, cc-by)
- Apollo (cc-by)
- Funders
- Biotechnology and Biological Sciences Research Council (1501548)
- Research Executive Agency (631609)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
- Keywords
- nanobodies, antibody binding sites, antibody-antigen binding, camelid single-domain antibodies, sequence variation, binding specificity
- MeSH terms
- Amino Acid Sequence, Animals, Antibody Specificity, Antigens, Models, Molecular, Protein Conformation, Crystallography, X-Ray, Single-Chain Antibodies
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