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Sci. Signal., 29 May 2012
Vol. 5, Issue 226, p. ra39
[DOI: 10.1126/scisignal.2002979]


Sequence-Specific Recognition of a PxLPxI/L Motif by an Ankyrin Repeat Tumbler Lock

Chao Xu1*, Jing Jin2*, Chuanbing Bian1, Robert Lam1{dagger}, Ruijun Tian2, Ryan Weist3,4, Linya You3,4, Jianyun Nie3,5, Alexey Bochkarev1, Wolfram Tempel1, Chris S. Tan2, Gregory A. Wasney1, Masoud Vedadi1, Gerald D. Gish2, Cheryl H. Arrowsmith1,6, Tony Pawson2,7, Xiang-Jiao Yang3,4, and Jinrong Min1,8{ddagger}

1 Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
3 Rosalind and Morris Goodman Cancer Research Center and Department of Biochemistry, McGill University, Montréal, Québec H3A 1A3, Canada.
4 Department of Medicine, McGill University Health Center, Montréal, Québec H3A 1A3, Canada.
5 The Third Affiliated Hospital, Kunming Medical College, Yunnan 650031, China.
6 Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
7 Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
8 Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

* These authors contributed equally to this work.

{dagger} Present address: Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.

Abstract: Ankyrin repeat family A protein 2 (ANKRA2) interacts with the plasma membrane receptor megalin and the class IIa histone deacetylases HDAC4 and HDAC5. We report that the ankyrin repeat domains of ANKRA2 and its close paralog regulatory factor X–associated ankyrin-containing protein (RFXANK) recognize a PxLPxI/L motif found in diverse binding proteins, including HDAC4, HDAC5, HDAC9, megalin, and regulatory factor X, 5 (RFX5). Crystal structures of the ankyrin repeat domain of ANKRA2 in complex with its binding peptides revealed that each of the middle three ankyrin repeats of ANKRA2 recognizes a residue from the PxLPxI/L motif in a tumbler-lock binding mode, with ANKRA2 acting as the lock and the linear binding motif serving as the key. Structural analysis showed that three disease-causing mutations in RFXANK affect residues that are critical for binding to RFX5. These results suggest a fundamental principle of longitudinal recognition of linear sequences by a repeat-type domain. In addition, phosphorylation of serine 350, a residue embedded within the PxLPxI/L motif of HDAC4, impaired the binding of ANKRA2 but generated a high-affinity docking site for 14-3-3 proteins, which may help sequester this HDAC in the cytoplasm. Thus, the binding preference of the PxLPxI/L motif is signal-dependent. Furthermore, proteome-wide screening suggested that a similar phosphorylation-dependent switch may operate in other pathways. Together, our findings uncover a previously uncharacterized sequence- and signal-dependent peptide recognition mode for a repeat-type protein domain.

{ddagger} To whom correspondence should be addressed. E-mail: jr.min{at}

Citation: C. Xu, J. Jin, C. Bian, R. Lam, R. Tian, R. Weist, L. You, J. Nie, A. Bochkarev, W. Tempel, C. S. Tan, G. A. Wasney, M. Vedadi, G. D. Gish, C. H. Arrowsmith, T. Pawson, X.-J. Yang, J. Min, Sequence-Specific Recognition of a PxLPxI/L Motif by an Ankyrin Repeat Tumbler Lock. Sci. Signal. 5, ra39 (2012).

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Modular evolution of phosphorylation-based signalling systems.
J. Jin and T. Pawson (2012)
Phil Trans R Soc B 367, 2540-2555
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