Structural basis for cyclic-nucleotide selectivity and cGMP-selective activation of PKG I.
Cyclic guanosine monophosphate (cGMP) and cyclic AMP (cAMP)-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here, we report that the PKG Iβ C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and we have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues that mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a "capping residue" for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation.
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Gilbert Y
GY
Huang
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Jeong Joo
JJ
Kim
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Albert S
AS
Reger
Department of Biochemistry, University of Kassel, Kassel 34132, Germany.
Robin
R
Lorenz
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Eui-Whan
EW
Moon
Department of Chemistry, Rice University, Houston, TX 77005, USA.
Chi
C
Zhao
Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Darren E
DE
Casteel
Department of Biochemistry, University of Kassel, Kassel 34132, Germany.
Daniela
D
Bertinetti
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON L8S 4M1, Canada.
Bryan
B
Vanschouwen
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON L8S 4M1, Canada.
Rajeevan
R
Selvaratnam
Rigaku Americas, The Woodlands, TX 77381, USA.
James W
JW
Pflugrath
Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Building 6R2100, Berkeley, CA 94720, USA.
Banumathi
B
Sankaran
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON L8S 4M1, Canada.
Giuseppe
G
Melacini
Department of Biochemistry, University of Kassel, Kassel 34132, Germany.
Friedrich W
FW
Herberg
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address: ckim@bcm.edu.
Choel
C
Kim
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D006152:Cyclic GMP / Q000737:chemistry* / Q000378:metabolism; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
D011994:Recombinant Proteins; D001120:Arginine; D000242:Cyclic AMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D007930:Leucine; D006152:Cyclic GMP