Structural basis for tankyrase‐RNF146 interaction reveals noncanonical tankyrase‐binding motifs

Abstract

Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.

TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2

“…The Tankyrase-binding motif is a loosely conserved motif of RxxDG . The first and last amino acid (R1 and G6) in peptides or fragments that span the Tankyrase-binding motif of 3BP2, Axin1, RNF146 and IRAP abolish the interaction with Tnks-1/2 and are considered to be invariant amino acids (DaRosa et al, 2018;Morrone et al, 2012;. Mutation analysis of a peptide spanning the TBD of 3BP2 demonstrated that at position 4, glycine, proline, alanine and cysteine are preferred while isoleucine, leucine and valine prevent Tnks-1/2 binding to the 3BP2 peptide ).…”

“…Tnks-1/2 both have a PARP domain, a sterile alpha motif (SAM) and 5 Ankyrin-repeat clusters (ARCs) (Citarelli et al, 2010). The SAM domain mediates self-polymerization of Tnks-1/2, which is a biophysical property required for Tnks-1/2 function (De Rycker and Price, 2004;De Rycker et al, 2003;Mariotti et al, 2016).The ARCs bind to a loosely conserved Tankyrase-binding motif (TBM) -RxxDG, where x represents any amino acid and  is a small hydrophobic amino acid or glycine -that is embedded in target proteins (DaRosa et al, 2018;Morrone et al, 2012;. A central role of Tnks-1/2 is in protein turnover by the UPS (Bhardwaj et al, 2017;Chang et al, 2003;Cho-Park and Steller, 2013;Huang et al, 2009;Levaot et al, 2011;Li et al, 2017b;Zhang et al, 2011).…”

Solution NMR assignment of the ARC4 domain of human tankyrase 2

“…Studies reported in 2011 and 2012 first revealed the structural basis of substrate recruitment by tankyrase (Guettler et al 2011 ; Morrone et al 2012 ). To date, numerous additional crystal structures of tankyrase ARCs from both TNKS and TNKS2, typically complexed with TBM peptides, are available (Li et al 2016 ; Eisemann et al 2016 ; Xu et al 2017a , b ; DaRosa et al 2018 ).…”

“…Additional contacts are provided by residues at TBM positions 4 and 5, which interact with a ‘central patch’ in the peptide-binding pocket, and at TBM position 8, where an acidic residue can confer increased binding affinity through a salt bridge (Guettler et al 2011 ). The eight amino acids of the TBM do not need to be contiguous: the essential arginine can be N-terminally displaced to varying extent, and structural plasticity or looping of such TBM peptides enables both the arginine and a hydrophobic side chain at position 4 to dock as in “canonical” TBM peptides (Morrone et al 2012 ; DaRosa et al 2018 ). Moreover, there is evidence for tankyrase targets devoid of detectable TBM sequences (Li et al 2017 ; Bhardwaj et al 2017 ).…”

Nuclear PARPs and genome integrity

“…The PAR ligand allosterically activates the E3 ligase through a conformational change in the N-terminal RING domain to promote K48-ubiquitination of its targets (DaRosa et al 2015). RNF146 can also bind to the ARC domains in tankyrase 1 or tankyrase 2 directly through motifs in its C terminus, thereby favoring PARylated tankyrases and their bound PARylated targets for ubiquitination and degradation (DaRosa et al 2015(DaRosa et al , 2018. In this way, tankyrases regulate the cellular levels of several disease-related cytoplasmic proteins including Axin, 3BP2, PTEN, and the angiomotins, which are key regulators of the Wnt/β-catenin, SRC, AKT, and Hippo signaling pathways, respectively (Huang et al 2009;Levaot et al 2011;Li et al 2015;Wang et al 2015).…”