6DF1

Anti-phosphotyrosine antibody 4G10-4D5 Fab complexed with phosphotyrosine peptide

Summary for 6DF1

• Entry DOI: 10.2210/pdb6df1/pdb
• Descriptor: Anti-phosphotyrosine antibody 4G10-4D5 heavy chain, Anti-phosphotyrosine antibody 4G10-4D5 light chain, LEU-PTR, ... (4 entities in total)
• Functional Keywords: antibody, recombination, immune system
• Biological source: Homo sapiens; More
• Total number of polymer chains: 3
• Total formula weight: 52588.97

Authors

Mou, K.,Leung, K.,Wells, J.A. (deposition date: 2018-05-13, release date: 2018-11-28, Last modification date: 2024-10-23)

Primary citation

Mou, Y.,Zhou, X.X.,Leung, K.,Martinko, A.J.,Yu, J.Y.,Chen, W.,Wells, J.A.
Engineering Improved Antiphosphotyrosine Antibodies Based on an Immunoconvergent Binding Motif.
J. Am. Chem. Soc., 140:16615-16624, 2018

PubMed Abstract: Phosphotyrosine (pY) is one of the most highly studied posttranslational modifications that is responsible for tightly regulating many signaling pathways in eukaryotes. Pan-specific pY antibodies have emerged as powerful tools for understanding the role of these modifications. Nevertheless, structures have not been reported for pan-specific pY antibodies, greatly impeding the further development of tools for integrating this ubiquitous posttranslational modification using structure-guided designs. Here, we present the first crystal structures of two widely utilized pan-specific pY antibodies, PY20 and 4G10. The two antibodies, although developed independently from animal immunizations, have surprisingly similar modes of recognition of the phosphate group, implicating a generic binding structure among pan-specific pY antibodies. Sequence alignments revealed that many pY binding residues are predominant in the mouse V germline genes, which consequently led to the convergent antibodies. On the basis of the convergent structure, we designed a phage display library by lengthening the CDR-L3 loop with the aid of computational modeling. Panning with this library resulted in a series of 4G10 variants with 4 to 11-fold improvements in pY binding affinities. The crystal structure of one improved variant showed remarkable superposition to the computational model, where the lengthened CDR-L3 loop creates an additional hydrogen bond indirectly bound to the phosphate group via a water molecule. The engineered variants exhibited superior performance in Western blot and immunofluorescence.

PubMed: 30398859
DOI: 10.1021/jacs.8b08402

Experimental method

X-RAY DIFFRACTION (2.3 Å)