Volume 22, Issue 12, Pages 1653-1661 (December 2015) Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα  Thomas M. Moon, Nathan R. Tykocki, Jessica L. Sheehe, Brent W. Osborne, Werner Tegge, Joseph E. Brayden, Wolfgang R. Dostmann  Chemistry & Biology  Volume 22, Issue 12, Pages 1653-1661 (December 2015) DOI: 10.1016/j.chembiol.2015.11.005 Copyright © 2015 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2015 22, 1653-1661DOI: (10. 1016/j. chembiol. 2015 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Structure of the cGMP Binding Sites from the Regulatory Domain of PKG Iα (A) Emphasis is given to the asymmetric interaction of helical segments between the cGMP binding sites (denoted B and B′) on opposing protomers of PKG Iα (PDB: 3SHR). Also shown are the switch helix domains (SW and SW′) from each protomer. (B) Directional view, indicated by arrow in (A), detailing the interaction of the knob residues (F350′, F351′, L354′) with the nest (N353′; not depicted). (C) Electrostatic surface representation of the cGMP B-site demonstrating its amino acid composition, the boundaries of the nest, and its location relative to the cGMP- binding site. The dashed line represents the demarcation of the regions of the nest and the cGMP-binding site within the B domain. Chemistry & Biology 2015 22, 1653-1661DOI: (10.1016/j.chembiol.2015.11.005) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Activation of PKG Iα by S-Tides (A and B) Activity of PKG Iα was measured using peptides with (A) C-terminal truncation modifications and (B) N-terminal truncation mutants derived from the parent compound S1.1. Activity is expressed as a percentage relative to 5 μM cGMP for individual experiments. Values indicated are the mean ± SD. (C and D) Secondary structure determination of (C) C-terminal and (D) N-terminal truncation mutants as measured by CD spectroscopy. Mean residue ellipticity (θ) is expressed as mdeg × cm2/dmol × residue. Chemistry & Biology 2015 22, 1653-1661DOI: (10.1016/j.chembiol.2015.11.005) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Effects of Peptide- or cGMP-Treated PKG Iα Added to the Cytosolic Face of Excised, Inside-Out Membrane Patches Containing KCa1.1 Channels (A) Representative control (left) and experimental traces (right) of single KCa1.1 channel openings in excised membrane patches from cerebral artery myocytes. (B) The combined results of single KCa1.1 recordings demonstrate a substantially increased NPo versus control for saturating cGMP levels and S-tides S1.1 and S1.5 (†p < 0.002, ∗p < 0.05). Mean values with SEM are represented. Dashed line indicates normalized channel activity from control patches. Chemistry & Biology 2015 22, 1653-1661DOI: (10.1016/j.chembiol.2015.11.005) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Intracellular Delivery of S-Tides by Reverse Permeablization Posterior cerebral arteries were subjected to reverse permeabilization with FITC-βAla2-S1.5 (experimental, left panel) and S1.5 (control, right panel) peptides. Following digestion using papain and collagenase, single smooth muscle cells were imaged using differential interference contrast microscopy (DIC) and confocal fluorescence microscopy. Chemistry & Biology 2015 22, 1653-1661DOI: (10.1016/j.chembiol.2015.11.005) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 5 S-Tides Cause BK Channel-Dependent Inhibition of Myogenic Tone Development in Posterior Cerebral Arteries (A) Diameter measurements for endothelium-denuded, reversibly permeabilized arteries demonstrating myogenic tone response in (Aa) control and (Ab) S1.5-treated arteries. (Ac–Ae) Summarized results of diameter measurements for endothelium-denuded arteries in control and permeabilized treatments are shown with values represented as the mean ± SEM. Differences in myogenic tone in response to pressure (Ac) as well as paxilline-induced constriction (Ad) are shown (∗p < 0.05). (B) Diameter measurements from endothelium-intact arteries for controls (Ba) and S1.5-treated arteries (Bb) show unaltered myogenic tone response (Bc) but small differences in paxilline-induced constriction of control and S1.5-treated arteries (Bd, ∗p < 0.02). For both sets of experiments, total constriction (Ae, Be) was unchanged between controls and S1.5-treated arteries. Chemistry & Biology 2015 22, 1653-1661DOI: (10.1016/j.chembiol.2015.11.005) Copyright © 2015 Elsevier Ltd Terms and Conditions