STRUCTURAL AND FUNCTIONAL STUDIES OF A NOVEL PHOSPHOTYROSINE-BINDING DOMAIN IN HAKAI, THE HYB DOMAIN, THAT TARGETS E-CADHERIN AND OTHER SRC SUBSTRATES

Abstract

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. We have discovered a new pTyr binding domain fold in protein called Hakai, an E3 ubiquitin ligase so named due to its role in E-cadherin ubiquitination and degradation (Hakai means destruction). We coined this new domain the HYB domain, which stands for Hakai pTyr-binding (HYB) domain. We showed that Hakai (amino acids 106-206) forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. This pTyr-binding domain bears a novel structure, with a completely unique fold, and lacks structural homology with any of the known structures in the Protein database. Moreover, sequence analysis shows that the HYB domain is found in approximately 70 species, thereby making it the third largest pTyr-binding domain after the SH2 and PTB domains. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features (Mukherjee M et al., 2012 EMBO J). Subsequently, we have characterized a novel dimeric switch of Hakai-truncated monomers during substrate recognition and describe the insights gained from solution studies and NMR structure. We have studied a C-terminal deletion mutant of the HYB domain (HYB-deltaC) comprising amino acids 106-194 that exists as a monomer in solution. The NMR structure revealed that this deletion mutant undergoes a dramatic structural change caused by a rearrangement of the atypical zinc-coordinated unit in the C-terminus of the HYB domain to a C2H2-like zinc finger. Moreover, using isothermal titration calorimetry, we show that a pTyr substrate peptide can induce dimerization of HYB-deltaC. This ligand-induced dimerization of is further validated. Overall, these observations suggest that the dimeric architecture of HYB domain is essential for the pTyr-binding property of Hakai.