Manjeet Mukherjee1,+, Soah Yee Chow2,+, Permeen Yusoff2, J. Seetharaman4, Cherlyn Ng2, Saravanan Sinniah2, Xiao Woon Koh2, Nur Farehan M. Asgar2, Dan Li2, Daniel Yim2, Rebecca A. Jackson2, Jingxi Yew2, Jingru Qian5, Audrey Iyu2, Yoon Pin Lim,3 Xingding Zhou1, Siu Kwan Sze5, Graeme R. Guy2* and J. Sivaraman1*
1 - Department of Biological Sciences, National University of Singapore
2 - Signal Transduction Laboratory, Institute of Molecular and Cell Biology
3 - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore
4 - X4 beamline, Brookhaven National Laboratory, USA
5 - School of Biological Sciences, Nanyang Technological University
+Joint first authors
Published in EMBO Journal, 17 January 2012. (Epub ahead of print.)
Phosphotyrosine-binding domains, typified by the SH2 and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (aa 106-206) revealed that it 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. 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.
Figure 1 Legend:
The Hakai dimer forms an intertwined, anti-parallel configuration spanning the points indicated in circles, with the entry and exit paths shown in green and brown arrows. The zinc-interacting side-chains are shown as green and brown sticks. Zinc-coordination takes place with the amino acid residues C172, H185 and H190 from one Hakai monomer and C166 from the other monomeric partner.
Figure 2 Legend: Immunoprecipitates of either wild-type Hakai or the Hakai zinc-coordinating mutants were tested for interaction with its endogenous substrate, cortactin. In the absence of Hakai dimerization with the zinc-coordinating mutants, the interaction with cortactin does not occur.
Figure 3 Legend: Six potential residues in Hakai were identified through NMR studies to interact with its substrate E-cadherin, and are highlighted as sticks in the ribbon representation of the crystal structure.