Volume 24, Issue 6, Pages 906-917 (June 2016) Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones  Renzo Johansson, Venkateswara Rao Jonna, Rohit Kumar, Niloofar Nayeri, Daniel Lundin, Britt-Marie Sjöberg, Anders Hofer, Derek T. Logan  Structure  Volume 24, Issue 6, Pages 906-917 (June 2016) DOI: 10.1016/j.str.2016.03.025 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Overall Structure of paNrdA (A) View of the asymmetric dimer from the side. The 2-fold symmetry axis of the dimer core is vertical. The two monomers of the core α/β barrel are colored in light and dark gray. The ATP cone domains ATPc1 and ATPc2 are light blue and dark blue in monomer A. In monomer B they are orange and red, respectively. The three dATP molecules bound to each monomer are shown as spheres. The same representation is used in (B) and (C). (B) View of the asymmetric paNrdA dimer from the top, looking down the dimer axis. (C) View of the paNrdA tetramer looking down the crystallographic 2-fold axis between the two dimers. (D) Modelling of the α4β2 complex, assuming that β2 binds in a near-productive complex approximately like the α2β2 model of (Uhlin and Eklund, 1994). The β2 dimer is shown in dark green. See also Figure S1. Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Solution SAXS Data for paNrdA (A) Superimposed scattering curves (merged data from three concentrations 0.5, 1.0, and 2.0 mg/ml) for the complexes with dATP (blue dots) and dGTP (red dots), as well as without nucleotides (purple dots) and the Δ147 mutant (gray dots). The curves are on an arbitrary scale and are shifted relative to each other on the y axis for clarity. (B) Scattering plot for dATP as in (A), with the calculated scattering from the crystal structure shown as a green line. The calculated scattering after EOM modelling of the N and C termini is shown in orange. (C) Superimposed P(r) functions for the dATP (blue) and dGTP (red) complexes, the apo protein (purple), the Δ147 mutant in the absence (gray dots) and presence (gray dotted line) of dATP. (D) The best ab initio reconstructions of the molecular envelopes of the dATP complex. The two panels are related by a 90° rotation around a horizontal axis. (E) Bead models for the dATP (blue) and dGTP (red) complexes superimposed. (F) Ab initio model for apo paNrdA. (G) Ab initio model for the Δ147 mutant in the presence of 0.5 mM dATP. For all bead models, the beads from DAMMIN were assigned van der Waals radii approximately equal to those used in DAMMIN, then a surface representation was made in PyMOL with a solvent radius of 3–4 Å to smooth the surface. In (F) and (G) a model for paNrdA with the first 147 residues removed has been fitted to the SAXS envelope for comparison. See also Tables S1–S3 and Figures S2–S6. Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Details of ATP Cone Domains 1 and 2 (A) Cartoon representation of ATPc1 colored from dark blue at the N terminus of the visible structure (residue 32) to red at the C terminus (residue 135). The two dATP molecules bound to ATPc1 are shown as sticks. The Mg2+ ion bound between the two dATPs is shown as a green sphere. |Fo| − |Fc| omit density for dATP and Mg2+ is shown as a blue mesh contoured at 3.0 σ. (B) Interactions of the two dATPs with ATPc1 and the Mg2+ ion. Side chain or main chain atoms interacting with the nucleotides are shown as sticks. For clarity the side chains of residues 43 and 44 are not shown. Residues from the NrdAg-type ATP cone motif are labelled in blue; residues involved in binding the second dATP are labelled red. (C) The NrdAg-type ATP cone binding one dATP molecule as exemplified by the a-site of the dATP-inhibited α4β4 complex of E. coli class I RNR (PDB: 4ERM). (D) ATP cone domains 1 and 2 in monomer B of paNrdA (light- and mid-blue, respectively) are superimposed. All side chains in the non-functional domain 2 are shown as thin lines. The first dATP molecule from ATP cone 1 is shown in stick representation. The red arrow highlights the inward collapse of the loop that normally binds the adenine base (red arrow). See also Table S4. Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 The Tetramerization Interfaces of paNrdA (A) Overview. The chains are colored as in Figure 1. The three main interaction areas between ATPc1 (blue/orange in the center of the panel) and itself or the C-terminal region of the core protein of two different monomers of the dimer (dark and light gray) are highlighted by colored boxes. (B) Details of the interactions between the two copies of ATPc1 in the tetramer. (C) Details of the second interface involving ATPc1 from monomer B (orange) and the core domain of monomer A from the second dimer (dark gray). (D) Details of the third interface involving ATPc1 from monomer A (light blue) and the core domain of monomer B from the second dimer (light gray). Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Characterization of the P. aeruginosa NrdA R119D Mutant with GEMMA, Enzyme Assays and Filter-Binding Studies (A–C) Scatchard plots showing the number of dATP-binding sites in the R119D (A), E106A/E126A (B), and H72A/D73A/Y830A mutants (C). The experiments were performed with 0.5 (●), 1.25 (■), and 2.5 (▴) μM of NrdA polypeptide. (D) Inhibition studies with dATP using R119D (■), E106/E126 (▴), H72A/D73A/Y830A (▾), and wild-type NrdA (●). Assays were performed with 0.3 μM of wild-type or mutant NrdA and 0.6 μM NrdB polypeptide in the presence of 0.7 mM 3H-CDP as substrate and 3 mM ATP together with increasing concentrations of dATP as allosteric effectors. (A–D) are based on three independent experiments with SEs shown. (E–G) GEMMA analysis of R119D (E), E106A/E126A (F), and H72A/D73A/Y830A (G). Each protein was at 0.5 μM in the absence or presence of 50 μM of the indicated allosteric effectors (with equimolar Mg2+ concentrations). The dotted lines represent the measured molecular masses of the monomer, dimer, and tetramer peaks (the theoretical monomer molecular mass is 107 kDa). Multiple experiments are fitted into each graph by adding a space of 500–2,000 units between the traces. Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Sequence Conservation of the ATP Cone Motifs and Degeneracy of ATP Cone Domain 2 (A) ATPc1 from paNrdA colored according to conservation within a set of 191 sequences representative of group NrdAz. Colors run from dark magenta for a ConSurf conservation score between 9 and 10, through white at 5 to light blue at 0–1. Residues with a score of 9–10 are shown as sticks and those with a score of 8–9 are shown as lines. The dATP molecules are shown as transparent “ghosts.” (B) A similar representation of ATP cone domain 2. (C) HMM profile for ATPc1. Blue dots indicate participation in the NrdAg-type ATP cone motif, red dots the motif responsible for binding the second dATP, black triangles the primary tetramerization interface and empty triangles the secondary interface. (D) HMM profile for ATPc2. (E) HMM profile for the ATP cone of the NrdAg subclass represented by E. coli NrdA. Blue dots indicate the residues involved in binding of the NrdAg-type dATP molecule (Figure 3C). Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Comparison of the ATP Cone Interactions Made in All Known Inhibited Complexes (A–F) In the left-hand panels (A), (B), and (C), the dATP-inhibited oligomeric complexes made by (A) P. aeruginosa NrdA; (B) E. coli NrdA and NrdB; and (C) yeast NrdA are shown in a surface representation. The α/β barrel cores in all complexes are colored light and dark gray, while the ATP cones involved in intermolecular interactions (ATPc1 in paNrdA) are colored light blue in one monomer and orange in the other. The non-functional ATPc2 cones in paNrdA are colored dark blue and red, respectively. In (B) the NrdB dimers are colored in two shades of green. Boxes indicate areas shown in more detail in (D), (E), and (F). In these panels the ATP cones making intermolecular interactions are shown in orange in approximately the same orientation in each panel. The ATP molecule(s) bound to the cone are shown as spheres. Structure 2016 24, 906-917DOI: (10.1016/j.str.2016.03.025) Copyright © 2016 Elsevier Ltd Terms and Conditions