Cation-induced intramolecular coil-to-globule transition in poly(ADP-ribose)

Abstract

Poly(ADP-ribose) (PAR), a non-canonical nucleic acid, is essential for DNA/RNA metabolism and protein condensation, and its dysregulation is linked to cancer and neurodegeneration. However, key structural insights into PAR's functions remain largely uncharacterized, hindered by the challenges in synthesizing and characterizing PAR, which are attributed to its length heterogeneity. A central issue is how PAR, comprised solely of ADP-ribose units, attains specificity in its binding and condensing proteins based on chain length. Here, we integrate molecular dynamics simulations with small-angle X-ray scattering to analyze PAR structures. We identify diverse structural ensembles of PAR that fall into distinct subclasses and reveal distinct compaction of two different lengths of PAR upon the addition of small amounts of Mg²⁺ ions. Unlike PAR₁₅, PAR₂₂ forms ADP-ribose bundles via local intramolecular coil-to-globule transitions. Understanding these length-dependent structural changes could be central to deciphering the specific biological functions of PAR.

Fig. 1. Biological functions of PAR and its relationship to chain length.

A ADP-ribosylation is heterogenous, presented as monomeric, linear or branched polymer forms as poly(ADP-ribose) or PAR. B PAR mediates various cellular processes at distinct subcellular locations. “Animal cell photo” by Tomáš Kebert & umimeto.org, used under CC BY-SA 4.0 and was cropped from the original image. C Pulldown of PAR-binding proteins in nuclear lysate coupled with mass spectrometry analysis indicates a preference of proteome to the length of PAR (data reproduced from Dasovich et al. with permission). D Biochemical studies showing that the strength of PAR-proteins interaction is influenced by the chain length (n). ‘−’ indicates no binding, ‘+’ indicates weak binding and ‘+++’ indicates strong binding (<1 µM dissociation constant). E PAR modulates the formation and dynamics of biomolecular condensates linked to human disease. In vitro microscopic studies showing that PAR chain length influences material properties of FUS condensates, both at 1 µM; scale bar, 5 µm. These images were adopted from Rhine et al. with permission.

Fig. 2. Molecular dynamics simulations of a 22-mer PAR polymer.

A Initial configuration of a typical simulation system. One 22-mer PAR polymer is placed in electrolyte solution (semitransparent molecular surface) containing 50 mM of NaCl. B End-to-end distance (R_EE, in red) and radius of gyration (R_g, in blue) of a PAR molecule as a function of time simulated in 50 mM NaCl electrolyte. C, E, G Average equilibrium end-to-end distance (circles) and radius of gyration (squares) of the 22-unit PAR polymer at various ion conditions. Dashed lines connect the points to guide the eye. Each data point represents a 250-ns trajectory average after exclusion of the first 50 ns in each simulation where the molecule started in an extended state. Error bars denote S.D. from the average value. D, F, H Representative snapshots of PAR conformation at the end of a 300 ns equilibration performed at the specified ion concentration conditions. The O3′, C3′, C4′, C5′, and O5′ atoms of PAR are shown in green, whereas all other atoms are in blue. Na⁺ (yellow), Cl⁻ (green) and Mg²⁺ (pink) ions located within 6 Å of PAR are shown as spheres. The ends of the PAR chains are depicted in red.

Fig. 3. Length-dependent collapse of PAR polymer.

A SAXS profiles of PAR₁₅ and PAR₂₂ in 100 mM NaCl with (red) and without (blue) the addition of 1 mM MgCl₂. Data are plotted in dimensionless Kratky axes, normalizing out size differences and emphasizing changes in shape and disorder in the mid-angle scattering regime. Experimental scattering is shown in light-colored points, and solid lines show molecular form factor (MFF) model fits to the data, extracting the Flory scaling parameter ν (Supplementary Fig. 3). Error bars are derived from experimental error and rebinning. B SAXS-derived R_g values for PAR₁₅ and PAR₂₂ in the conditions assayed. Error bars show errors in the linear Guinier fits used to extract R_g. (Supplementary Fig. 4) C Radius of gyration of PAR₁₅ and PAR₂₂ polymers in MD simulations carried out at 100 mM NaCl, with and without 1 mM MgCl₂. The histograms next to the timeseries plots illustrate the distribution of the R_g values. D Average simulated radius of gyration of PAR₁₅ and PAR₂₂ determined as a weighted mean ± square root of the weighted variance of the two Gaussian fit to the histograms. SAXS source data are provided as a Source Data file.

Fig. 4. Determining structural ensembles for PAR using MD and SAXS.

As an example, the case for PAR₁₅ in 100 mM NaCl is shown. The same plots for PAR₂₂ are shown in Supplementary Figs. 5, 6. A The pool of structures from the entire MD simulation is shown in orange, and the subset ensemble that agrees with the SAXS data in blue. Structures are parameterized in {R_g,R_EE} space. 1D histograms are weighted by the prevalence of each structure in the final ensembles. B Final agreement of the structural ensemble determined by EOM (blue) to the SAXS data (gray), compared to initial agreement of the structural ensemble of all MD conformers (orange). Gray error bars represent experimental errors. Residuals are shown in the bottom plot. C Ensembles of EOM-determined PAR structures, with and without Mg²⁺ for PAR₁₅ and PAR₂₂. Arrows show differential shifts to more compact states with the addition of Mg²⁺. D Tortuosities and E Fraction of adenine bases that are stacked in each structural ensemble of PAR, calculated as a weighted mean across the ensemble. For the tortuosity box-and-whisker plot in (D), the center mark is the medium and the box edges are the 25th and 75th percentiles; points outside the whisker edges are outliers (>2.7 S.D. from the mean). To gauge differences between groups, a two-sample two-sided t-test assuming unequal variances was performed, and ‘*’ denotes p < 0.05. Exact p-values: PAR₁₅ 100 mM NaCl vs PAR₁₅ 100 mM NaCl + 1 mM MgCl₂: p = 0.1862, PAR₂₂ 100 mM NaCl vs PAR₂₂ 100 mM NaCl + 1 mM MgCl₂: p = 5.969E-7, PAR₁₅ 100 mM NaCl vs PAR₂₂ 100 mM NaCl: p = 0.0027, PAR₁₅ 100 mM NaCl + 1 mM MgCl₂ vs PAR₂₂ 100 mM NaCl + 1 mM MgCl₂: p = 7.707E-11. For the base stacking plot in E, error bars show standard error. Number of structures in each refined ensemble: PAR15, 100 mM NaCl: 225; PAR15, 100 mM NaCl + 1 mM MgCl₂: 563; PAR22, 100 mM NaCl: 75; PAR22, 100 mM NaCl + 1 mM MgCl₂: 78. Note that, while the PAR15 pools have more unique structures, the weights of each structure are higher in the PAR22 pools (weights sum to 1000 in all pools).

Fig. 5. Backbone structural features of PAR₁₅ vs PAR₂₂ identified through spectral clustering.

A–D PAR₁₅ and PAR₂₂ in 100 mM NaCl, both without and with the presence of 1 mM MgCl₂, is shown. Top plots show the graphs of PAR structures in each ensemble, color-coded by the clusters identified by K-means. The box colors around each identified subclass match their locations in the graphs. Wireframe models show the mean PAR backbone conformation in each case—each dot represents the mean position of each pair of phosphorus atoms across the entire subclass, colored by the degree of spatial variance present across that class. Red squares denote the 1″ ends of the backbones and red triangles denote the 2′ ends. The fraction of each structural subclass within the entire ensemble is shown adjacent to the respective averaged backbone conformer models. E, F Proposed model of a critical length for coil-to-globule transitions in PAR in the presence of MgCl₂, linking to previously observed differences in binding and condensing certain proteins. Above a certain length, potentially between 15 and 22 subunits, PAR forms ADP-ribose bundles that impose super-anion functionality, accumulating negative charge and giving PAR a disproportionate amount of electrostatic potential. In longer PAR chains, these bundles may periodically appear along the chain, similar to the beads-on-a-string model of classical polymer theory.

Fig. 6. Polymeric differences of PAR₁₅ vs poly-adenine RNA (rA₃₀) in 100 mM NaCl.

A SAXS-derived R_g of PAR₁₅ vs rA₃₀. Error bars represent errors in the Guinier fits. B Mean fraction of adenine bases that are stacked in the PAR₁₅ and rA₃₀ structural ensembles. C Ensemble-averaged orientation correlation functions of PAR₁₅, compared to that of rA₃₀. D Mean correlation lengths of PAR₁₅ vs rA₃₀, computed across the structural ensembles. E Four structures from the conformational ensemble of PAR₁₅ that are most highly selected by EOM. F Four representative rA₃₀ structures; accessible via SASDFB9 in the Small Angle Scattering Biological Data Bank. Throughout this figure, blue refers to PAR₁₅, and green refers to rA₃₀. For (B–D), Error bars represent the variance in the datasets and are derived from analysis of N = 20 poly-A RNA structures (constituting the pool of structures from SASDFB9) and N = 225 PAR₁₅ structures (constituting the pool of structures in the current study).