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Updated: Aug 15, 2026

Bimolecular Fluorescence Complementation
Published on: April 15, 2011
Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of
In-Ja L Byeon1, Hongyuan Li, Haiyan Song
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.
Abstract:
The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226-269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226-269(3P), revealing not only local recognition of pThr234 but also the extension of the beta-sheet of the FHA domain by the addition of a beta-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.
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