SignificanceNegative feedback regulation of ribonucleotide reductase (RNR) activity by dATP is important for maintaining balanced intracellular 2ʹ-deoxynucleoside triphosphate (dNTP) pools essential for the high fidelity of DNA replication and repair. To date, this type of allostery has been nearly universally associated with dATP binding to the N-terminal ATP-cone domain of the class Ia RNR large subunit (canonical α2), resulting in an altered quaternary structure that is unable to productively bind the second subunit (β2). Here, we report our studies on activity inhibition by dATP of the Bacillus subtilis class Ib RNR, which lacks a traditional ATP-cone domain. This unprecedented allostery involves deoxyadenosine 5′-monophosphate (dAMP) binding to a newly identified site in a partial N-terminal cone domain, forming an unprecedented noncanonical α2.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu and Glu in β2 and Arg and Arg in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (FY) replacing the stable Tyr in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr-β2), 0.5 eq of dCDP/FY, and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed.

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (MnIII2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted MnIII2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min–1 mg–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from MnIII2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β2. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ∼1250 nmol min–1 mg–1 using a 1:1 mixture of NrdE:MnIII2-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (∼1 μM), NrdE is a monomer (α) and MnIII2-Y· NrdF is a dimer (β2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.

Many patients of choroidal neovascularization (CNV) are unresponsive to the current anti-VEGF treatment. The mechanisms for anti-VEGF resistance are poorly understood. We explore the unique property of the apolipoprotein A-I (apoA-I) binding protein (AIBP) that enhances cholesterol efflux from endothelial cells and macrophages to thereby limit angiogenesis and inflammation to tackle anti-VEGF resistance in CNV. We show that laser-induced CNV in mice with increased age showed increased resistance to anti-VEGF treatment, which correlates with increased lipid accumulation in macrophages. The combination of AIBP/ apoA-I and anti-VEGF treatment overcomes anti-VEGF resistance and effectively suppresses CNV. Furthermore, macrophage depletion in old mice restores CNV sensitivity to anti-VEGF treatment and blunts the synergistic effect of combination therapy. These results suggest that cholesterol-laden macrophages play a critical role in inducing anti-VEGF resistance in CNV. Combination therapy by neutralizing VEGF and enhancing cholesterol removal from macrophages is a promising strategy to combat anti-VEGF resistance in CNV.