Co-crystal structures of PKG Iβ (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding

Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate mo...

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Published in:	PloS one 2011-04, Vol.6 (4), p.e18413-e18413
Main Authors:	Kim, Jeong Joo, Casteel, Darren E, Huang, Gilbert, Kwon, Taek Hun, Ren, Ronnie Kuo, Zwart, Peter, Headd, Jeffrey J, Brown, Nicholas Gene, Chow, Dar-Chone, Palzkill, Timothy, Kim, Choel
Format:	Article
Language:	eng
Subjects:	Amino Acid Sequence Amino acids Anchoring Angina Angina pectoris Apoproteins - chemistry BASIC BIOLOGICAL SCIENCES Binding Binding Sites Biochemistry Biology Calorimetry Cloning Configurations Coronary vessels Crystal structure Crystallography Crystallography, X-Ray Cyclic AMP Cyclic AMP - chemistry Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - chemistry Cyclic GMP Cyclic GMP - chemistry Cyclic GMP-Dependent Protein Kinase Type I Cyclic GMP-Dependent Protein Kinases - chemistry Drugs Erectile dysfunction Genomics Guanine Humans Kinases Laboratories Ligands Medicine Memory Models, Molecular Molecular biology Molecular Sequence Data Nitric oxide Pain perception Pharmacology Phosphates Phosphodiesterase Phosphodiesterase inhibitors Protein Binding Protein kinase Protein Structure, Secondary Protein Structure, Tertiary Proteins Receptors Selectivity Signal transduction Signaling Smooth muscle Structural Homology, Protein Substrates Threonine Titration Titration calorimetry
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Summary:	Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.
ISSN:	1932-6203 1932-6203