Crystal Structure of cGMP-dependent Protein Kinase Iβ Cyclic Nucleotide-binding-B Domain: Rp-cGMPS Complex Reveals an Apo-like, Inactive Conformation

Campbell JC, et al., 591(1):221-230, FEBS Lett., 2017

The R-diastereomer of phosphorothioate analog of cGMP (Rp-cGMPS) is a well known inhibitor of cGMP-dependent protein kinase I (PKG I). However, no structural information is currently available to fully understand its mechanism of inhibition. Described herein is the determination of the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B): Rp-cGMPS complex. The binding affinity of Rp-cGMPS and cGMP towards PKG Iβ CNB-B was studied by Surface Plasmon Resonance (SPR) using a Pall ForteBio Pioneer FE instrument equipped with HisCap sensor chips. His-tagged PKG Iβ CNB-B was immobilized onto activated HisCap sensor chips via standard amine coupling chemistry. Subsequently, cGMP and RpcGMPS samples at different concentrations were injected in triplicates over the sensor chip using OneStepTM injections. SPR data obtained were fitted to a mass transport limited model using QDAT software. The equilibrium dissociation constants (KD) for the binding of Rp-cGMPS and cGMP to PKG Iβ CNB-B were determined. Collectively, the crystal structure and NMR data suggest that Rp-cGMPS inhibits PKG I by stabilizing an apo-like, inactive conformation of CNB-B.

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