Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design

Abstract

High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP.

Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)

“…Indeed, the C-terminal cyclic nucleotide-binding domain (CNB-B) of PKG I␤ was previously found to exhibit greater binding affinity for cGMP, which has been attributed to specific interactions between the base-binding region (BBR) 5 of CNB-B and the cGMP base moiety, as observed in previously solved crystallographic x-ray structures ( Fig. 1e) (17,18). However, the intracellular concentration of cAMP is typically significantly greater than that of cGMP (19 -23), thus potentially allowing cAMP to bind to PKG despite its lower binding affinity and suggesting that other means of cyclic nucleotide selectivity must be present in PKG.…”

“…Such partial agonism of cAMP is not fully explained by previously solved structures of PKG I␤, as they reveal very similar structural rearrangements of both CNB domains in response to either cAMP or cGMP binding (Fig. 1, b-d) (2,17,18) that are analogous to structural rearrangements observed for other CNB domains (8, 12, 24 -29). Hence, we hypothesize that the partial agonist response of PKG I␤ to cAMP may reflect differences in the dynamics of cAMP versus cGMP-bound PKG I␤.…”

“…This domain is adjacent to the catalytic domain (Fig. 1a), and it has been previously identified as a critical control unit for auto-inhibition and cGMP selectivity in PKG I␤ (17,18). In addition, we examined the CNB-B domain because although the CNB-A domain contributes to the cyclic nucleotide modulation of PKG I function (17,(35)(36)(37), the CNB-A domain undergoes only minor structural changes upon cyclic nucleotide binding (2,17).…”

Neutron crystallography aids in drug design

“…Furthermore, as neutrons (of the energies used for crystallographic experiments) are a non-destructive probe, the resulting structures are free from radiation damage even at room temperature (Blakeley et al, 2015). Knowledge of H-bonding networks, water molecule orientations and protonation states, along with details of hydrophobic and electrostatic interactions, can prove vital towards a better understanding of many biological processes, such as enzyme mechanisms (Casadei et al, 2014;Vandavasi et al, 2016) and ligand binding (Howard et al, 2016), and can help guide structure-based drug design (Huang et al, 2014).The first neutron crystallography study of a clinically used drug bound to its target was that of acetazolamide (AZM), a sulfonamide, which binds with high affinity to human carbonic anhydrase isoform II (Fisher et al, 2012). Human carbonic anhydrases (hCA) are zinc metalloenzymes that catalyze the interconversion of CO 2 and H 2 O to HCO 3 À and H + , an important reaction for many physiological processes including respiration, fluid secretion and pH regulation.…”

“…Furthermore, as neutrons (of the energies used for crystallographic experiments) are a non-destructive probe, the resulting structures are free from radiation damage even at room temperature (Blakeley et al, 2015). Knowledge of H-bonding networks, water molecule orientations and protonation states, along with details of hydrophobic and electrostatic interactions, can prove vital towards a better understanding of many biological processes, such as enzyme mechanisms (Casadei et al, 2014;Vandavasi et al, 2016) and ligand binding (Howard et al, 2016), and can help guide structure-based drug design (Huang et al, 2014).…”