Supplementary Figure 2: C-di-GMP binding to BcsA.
From: Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP
(a) An intercalated homodimer of c-di-GMP binds to BcsA's PilZ domain. An unbiased SigmaA-weighted mFo-DFc difference electron density for c-di-GMP contoured at 4σ is shown as a magenta mesh. The density was calculated after refining the protein structure and before placing any ligands. The c-di-GMP dimer is shown in sticks colored light blue or pale brown for the carbon atoms, respectively. (b) BcsA's PilZ domain tightly coordinates a c-di-GMP dimer, c-di-GMP-A and –B. One guanylate group of c-di-GMP-A (GA1) packs its guanine group into a pocket on the β-barrel surface formed by the conserved Gly614 and Gly670 where it is further stabilized by side chain interactions with Asp609 of the “DxSxxG” motif as well as Ser613 and Arg616. The guanine interacts with Asp609 via its cyclic N1 atom and exocyclic amine group and its carbonyl oxygen contacts the guanidinium group of Arg616. Ser611 of the “DxSxxG” motif does not directly contact GA1, however, it is likely that its interaction is mediated by an unresolved water molecule. The 2' hydroxyl of the GA1 ribose interacts with the hydroxyl group of Ser613. Arg584 of the TM8-β-barrel linker stacks on top of the GA1 guanine and forms a salt bridge with the phosphate group belonging to the second guanylate of c-di-GMP-A (GA2). The side chain of the invariant Arg580 of the TM8-β-barrel linker is co-planar with the guanine of GA2 and forms hydrogen bonds via its guanidinium group with the GA2's guanine N7 and carbonyl oxygen. Similar to the stacking interactions observed for GA1, the preceding Arg579 stacks on top of the GA2 guanine group. The ring N1 and exocyclic amine group of GA2 interact with the phosphate moiety of the second c-di-GMP molecule (c-di-GMP-B). C-di-GMP-B makes fewer interactions with BcsA and is primarily stabilized by c-di-GMP-A and residues belonging to the TM8-β-barrel linker. Its first guanylate closest to the β-barrel surface (GB1) forms π-π stacking interactions with the guanine of GA2 and hydrogen bonds via its ring N7 and carbonyl oxygen with Arg584, the same residue that stacks on top of the GA1 guanine. As observed for the guanine group of GA2, its ring N1 and exocyclic amine contact the phosphate group of the other c-di-GMP molecule, thereby stabilizing the intercalated c-di-GMP dimer. The second guanylate of c-di-GMP-B (GB2) interacts via its ring carbonyl oxygen with the backbone nitrogen as well as the Nɛ of the co-planar Arg579 and via the guanine's N1 and exocyclic amine with the invariant Gln578 of the TM8-β-barrel linker. In addition, its phosphate group forms a salt bridge with Arg580 that is co-planar with the guanine of GA2.
