Abstract
Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity.
Highlights
Human tankyrase-1 (TNKS) is a member of the poly(ADPribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose
Within the 8-amino acid– binding footprint, the TNKS-binding motifs (TBMs) of telomeric repeat-binding factor 1 (TRF1) and GMD differ at four positions: 3, 4, 7, and 8 (Fig. 1B, middle)
Substituting Ala for Arg at position 1 of the TBM (R13A in TRF1; R12A in GMD) effectively disrupted the pulldown interactions, consistent with studies illustrating the importance of R1 [15, 18]
Summary
TRF1 and GMD are both soluble constitutive homomultimers with TBMs at their extreme N termini (Fig. 1B). A histidine-tagged version of the ankyrin repeat region (TNKS-12345, residues 174 – 961), the minimal domain that is necessary and sufficient for binding partner interactions, was mixed with untagged fulllength constructs of TRF1 and GMD. Mutations to the TBMs of both TRF1 (TRF1(R13A)) and GMD (GMD(R12A)) abrogated their ability to compete with FITC-Axin for binding to TNKS-12345 (Fig. 2, C and D) Both TRF1 and GMD exhibited more efficient competition than their corresponding TBM peptides alone, suggesting that their quaternary structures present TBMs in orientations that allow them to simultaneously engage ARCs in ways that a single peptide cannot. We surmised that the competitive efficiency of TRF1 and GMD is a composite of the individual TBM affinities, and the total number and relative accessibility of TBMs are determined by quaternary structure
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