While the basis for the cyclic-nucleotide Tasocitinib specificity for PKG I has been previously studied, the exact molecular mechanism is not known. Because cGMP and cAMP are structurally different at only the 2-, 6-, and N1-positions of their purine rings, different amino acid contacts at these positions were proposed to mediate the specificity. Due to rotation around their glycosidic bonds, cyclic nucleotides exist in equilibrium between syn and anti conformations, with cGMP and cAMP favoring syn and anti conformations respectively. The cGMP-binding site of PKG and CNG PB 203580 channels has a threonine residue distinct from the cAMP receptors, and previous models based on the known structures of PKA and HCN channels have predicted that the hydroxyl group of these threonine residues interacts with the guanine 2-NH2 group of syn-cGMP through hydrogen bonds. We attempted to crystallize several CNBD-A and CNBD-A/B domains of PKG I, based on the previously solved crystal structures of PKA RIa. So far, only the CNBD-A corresponding to PKG Ib has yielded good diffraction quality crystals. In all, we obtained three crystal forms and solved eight molecules of PKG Ib, bound to a phosphate ion, cAMP or cGMP. Our structures explain some past biochemical observations on PKG I. One study demonstrated that intrachain disulphide bond formation between PKG Ia Cys117 and Cys195 activates the kinase. Consistent with this observation, the crystal structure of CNBD-A clearly shows that these residues are within the proper distance to form a disulphide bond upon oxidation. These residues are located within the A- and B-helices, and in analogy to PKA, the B-helix is expected to form contacts with the catalytic domain. We speculate that disulphide bond formation between these residues alters the conformation of the B-helix such that it no longer forms a binding surface for the catalytic domain. Another study demonstrated that cGMP-binding protected full-length PKG Ia from cleavage by chymotrypsin at Met200. Our structure reveals that this methionine links the B-helix to the PBC through hydrophobic interactions. It appears that cGMP-induced stabilization of the PBC would provide a stable hydrophobic interaction surface for the methionine, providing a possible explanation for the observed protection. A direct comparison between the three structures of the PKG Ib CNBD-A in the presence and absence of cyclic nucleotides, as well as with the homologous domain of PKA, provides a possible mechanism for cyclic nucleotide binding. In the absence of cyclic nucleotides, the conformation of CNBD-A is similar to the cyclic- nucleotide bound forms; with the exception of the b4/b5 strands which are in an open conformation with respect to PBC, as seen in the PO4 bound structure.