Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar 27;57(12):1833-1837.
doi: 10.1021/acs.biochem.8b00010. Epub 2018 Mar 13.

Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator

Affiliations

Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator

Oksana Gerlits et al. Biochemistry. .

Abstract

As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iβ:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

PubMed Disclaimer

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Interactions between the PKG II CNB-B domain and 8-pCPT-cGMP. (A) 8-pCPT cGMP binding pocket. CNB-B is colored light teal excluding the PBC and the αC helix, which are colored yellow and red, respectively. 8-pCPT-cGMP is shown as sticks and colored by atom type (carbon, yellow; nitrogen, blue; oxygen, red; phosphorus, orange). Key cGMP-interacting residues are colored by atom type with carbon colored black. O···O and O···N distances are shown as dashed lines with units of angstroms. The 2FOFC nuclear density map (blue mesh) is shown at 1.1σ for bound 8-pCPT-cGMP. A close-up view shows different side chain conformations of Lys347 seen in the RT XN and LT X-ray structures with 8-cCPT-cGMP bound and the LT X-ray structure with cGMP bound. The LT X-ray structure bound with cGMP (PDB entry 5BV6) is colored blue. The LT X-ray structure bound with 8-pCPT-cGMP (PDB entry 5JIZ) is colored magenta. (B) Crystal contacts in the RT XN structure of PKG II CNB-B:8-pCPT-cGMP between Arg415 and Glu292(sym). The side chains of Arg415 and Glu292(sym) form a salt bridge. PKG II CNB-B domains are shown with a transparent surface, with the molecule at the origin colored gray and the symmetry-related molecule colored tan.
Figure 2
Figure 2
Structural changes of the PKG II CNB-B domain associated with 8-pCPT-cGMP binding. Superposition of the XN PKG II CNB-B:8-pCPT-cGMP and LT X-ray PKG II CNB-B:cGMP complexes. The XN PKG II:8-pCPT-cGMP is shown with the same color theme as in Figure 1. Key binding residues are shown as sticks. Both termini are labeled. The LT X-ray structure is colored gray.
Figure 3
Figure 3
Backbone amide H/D exchange patterns in (A) PKG Iβ:cGMP and (B) PKG II:8-pCPT-cGMP complexes. N- and C-termini are labeled. Fully exchanged amides are colored gray, partially exchanged amides blue, and nonexchanged amides red. Proline residues are colored green. Close-up views show key binding residues.
Figure 4
Figure 4
8-pCPT-cGMP binding pocket. The surface of the binding pocket is colored red. The binding pocket was calculated using Hollow.
Scheme 1
Scheme 1
Chemical Diagrams of cGMP and 8-pCPT-cGMP

Similar articles

References

    1. Francis SH, Busch JL, Corbin JD, Sibley D. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev. 2010;62:525–563. - PMC - PubMed
    1. Feil R, Lohmann SM, de Jonge H, Walter U, Hofmann F. Cyclic GMP-Dependent Protein Kinase and the Cardiovascular System. Insights from Genetically Modified Mice. Circ Res. 2003;93:907–916. - PubMed
    1. Butt E, Geiger J, Jarchau T, Lohmann SM, Walter U. The cGMP-dependent protein kinase: gene, protein, and function. Neurochem Res. 1993;18:27–42. - PubMed
    1. Hofmann F, Bernhard D, Lukowski R, Weinmeister P. cGMP-regulated protein kinases (cGK) Handb Exp Pharmacol. 2009;191:137–162. - PubMed
    1. Reed RB, Sandberg M, Jahnsen T, Lohmann SM, Francis SH, Corbin JD. Fast and Slow Cyclic Nucleotide-dissociation Sites in cAMP-dependent Protein Kinase Are Transposed in Type 1β cGMP-dependent Protein kinase. J Biol Chem. 1996;271:17570–17575. - PubMed

Publication types

Substances