Structural and Biochemical Insights into the Arabinose and Xylose Derivatives of Cyclic-GAMP-Mediated Inhibition of Mycobacterium tuberculosis Cyclic-di-AMP Phosphodiesterase

Abstract Bacterial infection, including that ofMycobacterium tuberculosis, leads to the accumulation of the bacterial cyclic dinucleotides (CDN), c-di-AMP and c-di-GMP, and the host cGAMP synthase-catalyzed CDN, 2′3′-cGAMP in the cytosol, which activates STING-dependent type I interferon (IFN) and NF-κB immune responses. The mycobacterial cyclic dinucleotide phosphodiesterase (CdnP) secreted into the host macrophages blunts host immunity by directly cleaving bacterial- and host-derived CDNs. The arabinose- and xylose-modified 2′3′-cGAMP (2′3′-(A/X)cGAMP) analogues act as potent STING agonists and resist hydrolysis by the host-PDE ENPP1. Here, we report that 2′3′-(A/X)-cGAMP analogues bind to CdnP and compete with its substrate binding. Further studies revealed that these analogues inhibit the catalytic activity of CdnP. The cocrystal structure demonstrates that the arabinose-derived 2′3′-cGAMP (AR-cGAMP) analogue is trapped in an unusual U-shaped conformation in the substrate-binding pocket, away from the catalytic residue and Mn2+, which suggests that CdnP is incompetent to hydrolyze the analogue and cannot accept the other CDN substrate for hydrolysis. Given that several bacterial and viral pathogens deploy CDN phosphodiesterase enzymes to hydrolyze both host and pathogen-derived STING agonists, sugar-modified CDNs can be used to weaken bacterial and viral defenses and stimulate the STING-mediated host immunity against these pathogens.