An endogenous dAMP ligand inBacillus subtilisclass Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP

Abstract

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.

Convergent Allostery in Ribonucleotide Reductase

“…Side-chain densities in the I-dimer interface ( Supplementary Fig. 4c) are modeled well by a previously reported crystal structure of the dAMP-bound I-dimer (PDB: 6CGL) 31 . A key feature of this interface is the "F47-loop" comprising residues 45-50, which immediately follows the partial ATP-cone sequence (Fig.…”

“…As ADP was not present in the crystallization condition, we suspect that it is a hydrolysis product of ATP (SI Methods). The bound ADP retains an H-bond to the α-phosphate and all of the adenine-specific interactions that were previously observed in dAMP-bound structures 31 ( Supplementary Fig. 6b 31 .…”

“…The bound ADP retains an H-bond to the α-phosphate and all of the adenine-specific interactions that were previously observed in dAMP-bound structures 31 ( Supplementary Fig. 6b 31 . In this position, R117 forms part of the I-dimer interface ( Supplementary Fig.…”

“…3a, orange circles). Likewise, SEC-SAXS of holo-NrdE with 1 mM ATP and 0.5 mM CDP yielded a single scattering component that is well described by the theoretical scattering of a monomer 31 ( Supplementary Fig. 5a).…”

Still the most interesting enzyme in the world

“…An unexpected feature of the P. aeruginosa enzyme is the presence of 2 bound dATP molecules on one ATP cone and none on the other (6). Further heterogeneity in ATP/dATP coarse control was seen recently with the class Ib RNR from Bacillus subtilis (7). The endogenous form of a noncanonical R1 of this enzyme was shown to bind dAMP; the dAMP was shown to interact with dATP to form catalytically inactive oligomers.…”