Structural Analysis of Tankyrase-1 Regulation and Binding Partner Interaction

Poly(ADP-Ribose) (PAR) is a transient posttranslational modification that is involved in the regulation of several critical cellular processes including DNA damage repair and Wnt signaling. PAR is synthesized by Poly(ADP-Ribose) Polymerases (PARPs), that utilize NAD+ as a substrate to modify themselves and target proteins with PAR (termed PARylation). PARPs possess conserved catalytic PARP (CAT) domains, however the unique regulatory domains of individual family members endow each PARP with their own unique function(s). Much is understood about the regulatory mechanism of founding family member PARP-1. However, although several biological functions have been identified for other family members, a gap persists in our understanding of their regulatory mechanisms. Tankyrase-1 (TNKS, or PARP-5A) interacts with a growing number of structurally and functionally unique binding partners utilizing a multivalent ankyrin repeat domain. Through these interactions TNKS regulates several critical processes including Wnt signaling, Golgi trafficking, telomere maintenance, and apoptosis, and inhibition of TNKS catalytic activity has applications in the treatment of fibrosis as well as colorectal and gastric cancer. Although binding is required for PARylation, some binding partners can engage TNKS without being modified. TNKS is therefore capable of promiscuous binding while simultaneously deciding which partners do and do not get PARylated. Unfortunately, binding partners demonstrate such remarkable heterogeneity that no pattern has yet emerged that could identify a unifying regulatory mechanism. Prior to the work presented here, the study of the ankyrin repeat domain had been restricted to small fragments, and only limited studies had been performed that analyzed TNKS interaction with full length binding partners. To better understand the factors that govern TNKS regulation, we utilized a multifaceted structural and biochemical approach to define TNKS as a binding platform, and elucidate the features of TNKS:binding partner interactions that are required for binding and PARylation. This study has provided the first structural analysis of the entire ankyrin repeat domain, as well as the first comprehensive analysis of TNKS interaction with full-length binding partners. Our data demonstrate that the ankyrin repeat domain is a heteromultivalent binding platform that can dynamically sample unique conformations in order to facilitate binding. Although this domain accommodates significant variation amongst its many binding partners, PARylated binding partner telomeric repeat binding factor 1 (TRF1) possesses unique sequence and structural features that are critical for TNKS interaction and PARylation. However, we also found that the same factors do not affect the mechanism that prevents modification of non-PARylated binding partner GDP-Mannose-4,6-Dehydratase (GMD), indicating that there are additional factors that can affect TNKS regulation. Our data thus highlight the need for further study of the TNKS:binding partner interface in order to identify additional parameters that govern interaction, and elucidate the mechanisms that drive TNKS regulation. Importantly, this study provides unique insights into the characteristics of the ankyrin repeat domain that allow TNKS to function as a master scaffolding protein, and regulate multiple cellular processes. We further identify critical aspects of TNKS:binding partner interaction that may provide novel targets for TNKS inhibition, potentially aiding in the realization of TNKS inhibition therapy in the treatment of human disease