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6w9g
From Proteopedia
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
Structural highlights
DiseaseCD40L_HUMAN Defects in CD40LG are the cause of X-linked immunodeficiency with hyper-IgM type 1 (HIGM1) [MIM:308230; also known as X-linked hyper IgM syndrome (XHIM). HIGM1 is an immunoglobulin isotype switch defect characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. Affected males present at an early age (usually within the first year of life) recurrent bacterial and opportunistic infections, including Pneumocystis carinii pneumonia and intractable diarrhea due to cryptosporidium infection. Despite substitution treatment with intravenous immunoglobulin, the overall prognosis is rather poor, with a death rate of about 10% before adolescence.[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] FunctionCD40L_HUMAN Mediates B-cell proliferation in the absence of co-stimulus as well as IgE production in the presence of IL-4. Involved in immunoglobulin class switching.[11] Release of soluble CD40L from platelets is partially regulated by GP IIb/IIIa, actin polymerization, and an matrix metalloproteinases (MMP) inhibitor-sensitive pathway.[12] Publication Abstract from PubMedGenetically 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 Ile(L)98 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 pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. 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. Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.,Henderson JN, Simmons CR, Fahmi NE, Jeffs JW, Borges CR, Mills JH Biochemistry. 2020 Sep 22;59(37):3401-3410. doi: 10.1021/acs.biochem.0c00474., Epub 2020 Sep 3. PMID:32845612[13] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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