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.

Ligand-binding domain subregions contributing to bimodal agonism in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand-binding domain (BD), and this binding typically increases channel open probability (P(o)) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g((10 mM cGMP))/g((3 mM cGMP)) < 0.75. When all of the BD sequence C terminal of strand ß6 was replaced, g((10 mM cGMP))/g((3 mM cGMP)) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved "phosphate-binding cassette" motif that contacts ligand; this produces high P(o) values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g((30 mM cGMP))/g((3 mM cGMP)) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.