Aufsatz in einer Fachzeitschrift
Structural Basis of Analog Specificity in PKG I and II
Details zur Publikation
Autor(inn)en: | Campbell, J.; Henning, P.; Franz, E.; Sankaran, B.; Herberg, F.; Kim, C. |
Publikationsjahr: | 2017 |
Zeitschrift: | ACS Chemical Biology |
Seitenbereich: | 2388-2398 |
Jahrgang/Band : | 12 |
Heftnummer: | 9 |
ISSN: | 1554-8929 |
eISSN: | 1554-8937 |
DOI-Link der Erstveröffentlichung: |
Zusammenfassung, Abstract
Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique pi/pi interaction through Arg285 at beta4 with the PET moiety, whereas the A site of PKG II has a larger beta5/beta6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.
Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique pi/pi interaction through Arg285 at beta4 with the PET moiety, whereas the A site of PKG II has a larger beta5/beta6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.
Schlagwörter
Cyclic GMP/analogs & derivatives/metabolism, Cyclic GMP-Dependent Protein Kinase Type I/chemistry/metabolism, Cyclic GMP-Dependent Protein Kinase Type II/chemistry/metabolism, EC 2.7.11.12 (Cyclic GMP-Dependent Protein Kinase Type I, EC 2.7.11.12 (Cyclic GMP-Dependent Protein Kinase Type II, H2D2X058MU (Cyclic GMP, Humans, Protein Conformation, Substrate Specificity, Thionucleotides, Thionucleotides/metabolism
