Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 116-124 |
| Number of pages | 9 |
| Journal | Structure |
| Volume | 22 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 7 2014 |
Bibliographical note
Funding Information:We thank K. Sippel, M. Zhou, C. Peters, C.J. Lim, A. Koyfman, and S.S. Taylor for critical reading of the manuscript. We also thank E. Franz (University of Kassel) for expert technical assistance, S.R. Wasserman (Eli Lilly Beamline, APS) for his assistance with data collection, and S. Badal and F. Danesh (Baylor College of Medicine [BCM]) for their assistance with HEK293T culture. C.K. is funded by National Institutes of Health (NIH) grant R01 GM090161 and a BCM seed grant. G.Y.H. is supported by the Houston Area Molecular Biophysics Program, National Institute of General Medical Science grant no. T32GM008280. G.M. is funded by the Canadian Institute of Health Research. F.W.H. is supported in part by European Union grant “Affinomics” (Contract 222635) and the Federal Ministry of Education and Research Project “NoPain” (FKZ0316177F). The Berkeley Center for Structural Biology is supported in part by the NIH, the National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under contract no. DE-AC02-05CH11231.