6c4u
From Proteopedia
Engineered FHA with Myc-pTBD peptide
Structural highlights
FunctionRAD53_YEAST Controls S-phase checkpoint as well as G1 and G2 DNA damage checkpoints. Phosphorylates proteins on serine, threonine, and tyrosine. Prevents entry into anaphase and mitotic exit after DNA damage via regulation of the Polo kinase CDC5. Seems to be involved in the phosphorylation of RPH1.[1] [2] [3] [4] [5] Publication Abstract from PubMedTranscription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLPpTPPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a KD value of 800nM and had >1,000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its beta4-beta5 and beta10-beta11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD's specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation. Generating a recombinant phosphothreonine-binding domain for a phosphopeptide of the human transcription factor, c-Myc.,Venegas LA, Kall SL, Bankole O, Lavie A, Kay B N Biotechnol. 2018 May 12. pii: S1871-6784(18)30120-1. doi:, 10.1016/j.nbt.2018.05.001. PMID:29763736[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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