NSRRC Activity Report 2022

048 NSRRC ACTIVITY REPORT 2022 The Pc STING and My STING dimers are similar and have an RMSD of 1.09 Å for 272 Cα pairs. The electron density maps unambiguously identified the bound CDN as cyclic- di-GMP (CDG; Figs. 1(c) and 1(d) ), which originates from the expression host and is copurified with the protein, as verified through mass spectroscopic analysis. Recognition of the CDG, bound in a symmetric manner to the dimeric protein, is dictated by interactions of the guanine base with the side chains of Arg233/Asp252 and the backbone of His238 in Pc STING (conversely, Arg230/Asp251 and His235 in My STING; Fig. 1(e) ). In addition, each nucleobase of the CDG is sandwiched by Phe171 of one Pc STING protomer and Tyr235 of the other Pc STING protomer, and there are extensive π–π stacking interactions (Leu169 and Tyr232 in My STING; Fig. 1(f) ). The aromatic tyrosine side chain also interacts with the planar guanidinium group of Arg233, creating a unique Phe/Gua/Tyr/Arg four-layer double-stack formation supported by the hydrogen bonds between Arg233/Asp252 and the CDG ( Fig. 1(g) ). The binding mode was challenged by mutating Tyr232 into Arg in My STING. The affinity of binding to CDG was measured using isothermal titration calorimetry and was revealed to be nearly 1200 times lower than in the wild type. The binding of CDG to the STING domain of full-length TIR-STING can induce TIR oligomerization into long filament and activate the NAD + cleavage activity of its TIR domain. The subsequent depletion of NAD + results in cell death. In fact, overexpression of wild-type Pc TIR-STING or My TIR-STING in Escherichia coli was discovered to result in considerable growth inhibition, and mutating the specificity determining residue Arg233 or Asp252 in Pc TIR-STING reversed this growth inhibition, thereby validating the amino acid’s vital roles in CDG recognition. Moreover, the oligomerization state is implicated in the crystal structures of both Pc STING and My STING. Each V-shaped dimer of Pc STING associates with its neighbors on both sides, which are symmetry-related through unit-cell translation along the c -axis ( Figs. 2(a) and 2(b) ). A similar arrangement of dimers along the a -axis of the My STING crystal suggests a common mode of dimer–dimer interaction ( Figs. 2(c) and 2(d) ). The interface excludes surface area of size 1440 Å 2 on the Pc STING dimer (1420 Å 2 on My STING), which is comparable to the subunit interface of 1120 Å 2 (1500 Å 2 ) in a dimer. If a continuous filament subsequently forms, each dimer has nearly 10% of its surface area buried, and at least 60 amino acid residues are involved ( Fig. 2(e) ). The oligomerization is stabilized by the highly complementary molecular shapes and electrostatic charges at the interface. In addition to large hydrophobic patches, specific interactions such as the salt bridges of Lys212-Asp301 and Lys220-Glu306 and the hydrogen bond of Ser213-Ser302 were observed in the Pc STING oligomer, and similar bonds were also present in My STING. However, mutations in these regions of My TIR-STING resulted in the aggregation of improperly formed oligomers. Fig. 1 : Structures of bacterial STING in a complex containing cyclic di-GMP. Surface and cartoon models of (a) Pc STING and (b) My STING dimers are presented, with colors differentiating the two protomers. The cyclic di-GMP is depicted as a space-filled model. The F o – F c maps for the ligand binding to (c) Pc STING and (d) My STING are contoured at the 4-σ level. The hydrogen bonding network and stacking interactions between Pc STING and cyclic di-GMP are depicted in (e) and (f) and further illustrated in a diagram (g). [Reproduced from Ref. 3]