Influence of chain length and branching on poly(ADP-ribose)–protein interactions

Abstract

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

Length-dependent Intramolecular Coil-to-Globule Transition in Poly(ADP-ribose) Induced by Cations

“…This distinction may align with PAR’s unique ability, compared to RNA, to bind positive ligands, both in 1:1 interactions and large-scale condensates. 4,17,18,22…”

“…This distinction may align with PAR's unique ability, compared to RNA, to bind positive ligands, both in 1:1 interactions and large-scale condensates. 4,17,18,22 Given that PAR does not form secondary structure and exists as a highly disordered homopolymer, it has historically presented challenges for structural characterization. The few published studies on PAR structure suggested that it lacks a well-defined structure but may have some subtle structural features.…”

“…1C, D). [16][17][18][19][20] In certain cases, such as with oncoprotein DEK, a threshold PAR length is necessary for appreciable binding to occur. 21 Moreover, 4-mer PAR does not induce the condensation of FUS (a key protein associated with multiple neurodegenerative diseases), whereas 8-and 16-mer do, and 32-mer drives its aggregation (Fig.…”

“…The binding affinity of PAR to proteins depends on their chain length on a global scale, where its specificity could be even down to a single ADP-ribose difference (Fig. 1C, D) [10][11][12][13][14] . In certain cases, such as with oncoprotein DEK, a threshold PAR length is necessary for appreciable binding to occur 15 .…”

Specific and shared biological functions of PARP2 – is PARP2 really a lil’ brother of PARP1?

“…63). PAR is an important interaction surface for DNA repair factors and other proteins; branching and the physical buildup of PAR plays key role in selecting PAR interacting partners (Refs 64, 65). It is important to note that PARP2 plays key role in generating branched PAR chains, while not influencing the number of ADP-ribose moieties in the chain (Ref.…”