Interaction of Ribonuclease III with the Regulatory Macrodomain Protein YmdB Analyzed by Docking Calculations and SPR Experiments

Ribonuclease III (RNase III) is a conserved bacterial endonuclease that cleaves double-stranded(ds) RNA structures and is essential in diverse RNA maturation and decay pathways [1,2]. RNase III is subject to multiple levels of regulation, allowing fine-tuning of its catalytic activity depending on the cellular physiological state. The regulatory macrodomain protein YmdB interacts with RNase III, and an increase in YmdB levels correlates with a decrease in RNase III activity in vivo [3]. However, the molecular details of the YmdB-RNase III interaction are not yet known. Here, docking calculations and computationally-driven mutagenesis were combined with surface plasmon resonance (SPR) experiments to identify energetically important determinants of the Escherichia coli YmdB-RNase III interaction. The computational results reveal two alternative YmdB binding sites in RNase III: one located in the N-terminal nuclease domain (RIIID) (also indicated by co-immunoprecipitation and chemical cross-linking [3]), and a novel site in the C-terminal dsRNA-binding domain (dsRBD). The binding site in the RIIID is composed of a cluster of negatively charged residues that interact with a conserved arginine in YmdB, and the importance of this interaction is confirmed by SPR analysis of the YmdB Arg to Ala mutation. These results suggest a mechanism of RNase III regulation in which YmdB can bind separate sites in a concentration-dependent manner, leading to inhibition of catalytic activity.[1] Nicholson A.W. 2014. Ribonuclease III mechanisms of double-stranded RNA cleavage. WIREs RNA 5:31-48.[2] Court D.L., Gan J., Liang Y.-H., Shaw G.X., Tropea J.E., Costantino N., Waugh D.S., Ji X. 2013. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 47:405-431.[3] Kim K.-S., Manasherob R., Cohen S.N. 2008. YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. Genes Dev. 22:3497-3508.