Domain organization of XAF1 and the identification and characterization of XIAP(RING) -binding domain of XAF1.
X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1(NTD) ) followed by a middle domain (XAF1(MD) ), a 42-residue unstructured linker and a C-terminal domain (XAF1(CTD) ). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAP(RING)  binding site (dissociation constant, K(d) , ∼18 μM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1(CTD)  and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAP(RING) -binding domain of XAF1 (XAF1(RBD) ) in the present study. We have also mapped the interaction sites for XAF1(RBD)  on XIAP(RING)  by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1(RBD) /XIAP interaction is essential for the ubiquitination of GST-XAF1(RBD)  fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1(RBD)  was found to be substantially ubiquitinated by XIAP(RING) . Base on these observations, we speculate a possible role of XAF1(RBD)  in targeting XAF1 for XIAP-mediated ubiquitination.
Department of Microbiology and State Key Laboratory for Emerging Infectious Diseases, University of Hong Kong, Hong Kong SAR, People's Republic of China.
Man Kit
MK
Tse
Chi Kong
CK
Cho
Wai Fung
WF
Wong
Bing
B
Zou
Sin Kam
SK
Hui
Benjamin Chun Yu
BC
Wong
Kong Hung
KH
Sze
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments; D010449:Peptide Mapping; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054875:Ubiquitination; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D051636:X-Linked Inhibitor of Apoptosis Protein; C417551:XAF1 protein, human; C496866:XIAP protein, human