Abstract
VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure–function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Muyldermans S (2020) Applications of nanobodies. Annu Rev Anim Biosci 9:401–421
Arbabi-Ghahroudi M (2017) Camelid single-domain antibodies: historical perspective and future outlook. Front Immunol 8:1589
Vincke C, Muyldermans S (2012) Introduction to heavy chain antibodies and derived nanobodies. Methods Mol Biol 911:15–26
Fang T, Duarte JN, Ling J et al (2016) Structurally defined αMHC-II nanobody-drug conjugates: a therapeutic and imaging system for B-cell lymphoma. Angew Chem Int Ed Engl 55:2416–2420
Henry KA, MacKenzie CR (2018) Editorial: single-domain antibodies – biology, engineering and emerging applications. Front Immunol 9:41
Yang EY, Shah K (2020) Nanobodies: next generation of cancer diagnostics and therapeutics. Front Oncol 10:1182
Akiba H, Tamura H, Kiyoshi M et al (2019) Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody. Sci Rep 9:15481
Henry KA, MacKenzie CR (2018) Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10:815–826
Gershoni JM, Roitburd-Berman A, Diman-Tov DD et al (2007) Epitope mapping: the first step in developing epitope-based vaccines. BioDrugs 21:145–156
Forsström B, Axnäs BB, Rockberg J et al (2015) Dissecting antibodies with regards to linear and conformational epitopes. PLoS One 10:e0121673
Toride King M, Brooks CL (2018) Epitope mapping of antibody-antigen interactions with X-ray crystallography. Methods Mol Biol 1785:13–27
White B, Huh I, Brooks CL (2019) Structure of a VHH isolated from a naive phage display library. BMC Res Notes 12:154
Quan S, Hiniker A, Collet J-F et al (2013) Isolation of bacteria envelope proteins. Methods Mol Biol 966:359–366
Dauter Z (2017) Collection of X-ray diffraction data from macromolecular crystals. Methods Mol Biol 1607:165–184
Evans P, McCoy A (2008) An introduction to molecular replacement. Acta Crystallogr D Biol Crystallogr 64:1–10
DiMaio F (2017) Rosetta structure prediction as a tool for solving difficult molecular replacement problems. Methods Mol Biol 1607:455–466
Liebschner D, Afonine PV, Baker ML et al (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877
Casañal A, Lohkamp B, Emsley P (2020) Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci 29:1069–1078
Read RJ, Adams PD, Arendall WB 3rd et al (2011) A new generation of crystallographic validation tools for the protein data bank. Structure 19:1395–1412
Laskowski RA, Jabłońska J, Pravda L et al (2018) PDBsum: structural summaries of PDB entries. Protein Sci 27:129–134
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ahmed, A.M., Brooks, C.L. (2022). X-ray Crystal Structure Analysis of VHH–Protein Antigen Complexes. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_26
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2075-5_26
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2074-8
Online ISBN: 978-1-0716-2075-5
eBook Packages: Springer Protocols