6W9G

Crystal Structure of the Fab fragment of humanized 5c8 antibody containing the fluorescent non-canonical amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine in complex with CD40L at pH 6.8

Summary for 6W9G

• Entry DOI: 10.2210/pdb6w9g/pdb
• Related: 1I9R 6BJZ 6W4W 6W5A
• Descriptor: CD40 ligand, 5c8* Fab (heavy chain), 5c8* Fab (light chain), ... (10 entities in total)
• Functional Keywords: immunoglobulin, cytokine, l-(7-hydroxycoumarin-4-yl)ethylglycine, immune system
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 9
• Total formula weight: 198468.31

Authors

Henderson, J.N.,Simmons, C.R.,Mills, J.H. (deposition date: 2020-03-23, release date: 2020-12-23, Last modification date: 2024-10-16)

Primary citation

Henderson, J.N.,Simmons, C.R.,Fahmi, N.E.,Jeffs, J.W.,Borges, C.R.,Mills, J.H.
Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.
Biochemistry, 59:3401-3410, 2020

PubMed Abstract: Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue Ile98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent ps of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in p. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.

PubMed: 32845612
DOI: 10.1021/acs.biochem.0c00474

Experimental method

X-RAY DIFFRACTION (1.82 Å)