Three-dimensional structure of a mycobacterial oligoribonuclease reveals a unique C-terminal tail that stabilizes the homodimer

Oligoribonucleases (Orns) are highly conserved DnaQ-fold 3′-5′ exoribonucleases that have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, that is, pGpG to GMP in several bacteria. Removal of pGpG is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can have feedback inhibition on phosphodiesterases, thereby perturbing cellular signaling pathways regulated by c-di-GMP. Perturbation of c-di-GMP levels not only affects survival under hypoxic, reductive stress, or nutrient-limiting conditions but also affects pathogenicity in infection models as well as antibiotic response in mycobacteria. Here, we have determined the crystal structure of MSMEG_4724, the Orn of Mycobacterium smegmatis (Ms_orn) to 1.87 Å resolution to investigate the function of its extended C-terminal tail that is unique among bacterial Orns. Ms_orn is a homodimer with the canonical RNase-H fold of exoribonucleases and conserved catalytic residues in the active site. Further examination of the substrate-binding site with a modeled pGpG emphasized the role of a phosphate cap and “3′OH cap” in constricting a 2-mer substrate in the active site. The unique C-terminal tail of Ms_orn aids dimerization by forming a handshake-like flap over the second protomer of the dimer. Our thermal and denaturant-induced unfolding experiments suggest that it helps in higher stability of Ms_orn as compared with Escherichia coli Orn or a C-terminal deletion mutant. We also show that the C-terminal tail is required for modulating response to stress agents in vivo. These results will help in further evaluating the role of signaling and regulation by c-di-GMP in mycobacteria.