8FTG

Biophysical and Structural Characterization of an Anti-Caffeine VHH Antibody

PDB DOI: https://doi.org/10.2210/pdb8FTG/pdb

Classification: IMMUNE SYSTEM
Organism(s): Lama glama
Expression System: Escherichia coli
Mutation(s): No

Deposited: 2023-01-12 Released: 2023-06-07
Deposition Author(s): Horn, J.R., Smith, C.A., Sonneson, G.J., Walter, R.
Funding Organization(s): National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.13 ?
R-Value Free: 0.164
R-Value Work: 0.142
R-Value Observed: 0.144

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.1 of the entry. See complete history.

Literature

Molecular recognition requires dimerization of a VHH antibody.

Smith, C.A., Sonneson, G.J., Hoey, R.J., Hinerman, J.M., Sheehy, K., Walter, R., Herr, A.B., Horn, J.R.

(2023) MAbs 15: 2215363-2215363

PubMed: 37243579 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1080/19420862.2023.2215363
Primary Citation of Related Structures:
8FTG

PubMed Abstract:
Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 ? resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

Organizational Affiliation

Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA.

Macromolecule Content

Total Structure Weight: 106.34 kDa
Atom Count: 8,398
Modelled Residue Count: 948
Deposited Residue Count: 968
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Anti-Caffeine VHH Antibody	A, B, C, D, E A, B, C, D, E, F, G, H	121	Lama glama	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 3 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
CFF (Subject of Investigation/LOI) Query on CFF Download Ideal Coordinates CCD File SDF format, chain I [auth B] SDF format, chain K [auth D] SDF format, chain M [auth F] SDF format, chain O [auth G] MOL2 format, chain I [auth B] MOL2 format, chain K [auth D] MOL2 format, chain M [auth F] MOL2 format, chain O [auth G] mmCIF format, chain I [auth B] mmCIF format, chain K [auth D] mmCIF format, chain M [auth F] mmCIF format, chain O [auth G]	I [auth B], K [auth D], M [auth F], O [auth G]	CAFFEINE C₈ H₁₀ N₄ O₂ RYYVLZVUVIJVGH-UHFFFAOYSA-N		Interactions Focus chain I [auth B] Focus chain K [auth D] Focus chain M [auth F] Focus chain O [auth G] Interactions & Density Focus chain I [auth B] Focus chain K [auth D] Focus chain M [auth F] Focus chain O [auth G]
TRS Query on TRS Download Ideal Coordinates CCD File SDF format, chain J [auth B] MOL2 format, chain J [auth B] mmCIF format, chain J [auth B]	J [auth B]	2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL C₄ H₁₂ N O₃ LENZDBCJOHFCAS-UHFFFAOYSA-O		Interactions Focus chain J [auth B] Interactions & Density Focus chain J [auth B]
CL Query on CL Download Ideal Coordinates CCD File SDF format, chain L [auth E] SDF format, chain N [auth F] SDF format, chain P [auth H] MOL2 format, chain L [auth E] MOL2 format, chain N [auth F] MOL2 format, chain P [auth H] mmCIF format, chain L [auth E] mmCIF format, chain N [auth F] mmCIF format, chain P [auth H]	L [auth E], N [auth F], P [auth H]	CHLORIDE ION Cl VEXZGXHMUGYJMC-UHFFFAOYSA-M		Interactions Focus chain L [auth E] Focus chain N [auth F] Focus chain P [auth H] Interactions & Density Focus chain L [auth E] Focus chain N [auth F] Focus chain P [auth H]