6df1
Anti-phosphotyrosine antibody 4G10-4D5 Fab complexed with phosphotyrosine peptide
Structural highlights
Publication Abstract from PubMedPhosphotyrosine (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. Engineering Improved Antiphosphotyrosine Antibodies Based on an Immunoconvergent Binding Motif.,Mou Y, Zhou XX, Leung K, Martinko AJ, Yu JY, Chen W, Wells JA J Am Chem Soc. 2018 Nov 19. doi: 10.1021/jacs.8b08402. PMID:30398859[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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