1. Volume 24, Issue 10, Pages 1629-1642 (October 2016)
Cyclic Purine and Pyrimidine Nucleotides Bind to the HCN2 Ion Channel and Variably Promote C-Terminal Domain Interactions and Opening 
Leo C.T. Ng, Igor Putrenko, Victoria Baronas, Filip Van Petegem, Eric A. Accili 
Structure 
Volume 24, Issue 10, Pages (October 2016)
DOI: /j.str
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2. Figure 1
Oligomerization of the HCN2 C-Linker/CNBD Is Promoted by cAMP, cGMP, and a Subset of Structurally Related Analogs
Plots of estimated average molecular weight versus concentration of purified HCN2 C-linker/CNBD protein are shown. The estimated molecular weight was determined by dynamic light scattering and is proportional to the population of oligomers in solution. An increase in oligomerization occurs with higher protein concentrations and further increased in the presence of cAMP, cGMP (left), cUMP (right), cPuMP, and 2-NH2-cPuMP (middle). Cyclic CMP (right) and cIMP (middle) have no effect on the apparent molecular weight. Values represent means ± SEM, determined from two separate preparations of purified protein measured on different days.
Structure  , DOI: ( /j.str )
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3. Figure 2
The Cyclic Purine Nucleotides cAMP and cGMP Bind to the HCN2 C-Linker/CNBD with Negative Cooperativity
(A) Chemical structure of the two ligands. The atoms in the purine ring are labeled for reference.
(B) Plots of heat and binding isotherms produced by progressive injections of cAMP (left) and cGMP (right) to 200 μM HCN2 C-linker/CNBD as indicated, measured by ITC.
(C) Bar graph showing the affinity and thermodynamics for the two binding events, a high-affinity and a low-affinity binding event, which were determined from the fit in (B). Values in the graph represent means ± SEM. Each mean was determined from independent ITC binding experiments as shown in Table 1; N in Table 1 refers to the number of independent experiments for each condition.
Structure  , DOI: ( /j.str )
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4. Figure 3
The Cyclic Pyrimidine Nucleotide cUMP, but Not cCMP, Binds to the HCN2 C-Linker/CNBD with Negative Cooperativity
(A) Chemical structure of the two ligands. The atoms in the pyrimidine ring are labeled.
(B) Plots of heat and binding isotherms produced upon progressive injections of 2 mM cCMP and cUMP to 200 μM HCN2 C-linker/CNBD measured by ITC.
(C) Bar graph showing the affinity and thermodynamics of binding, which were determined from the fit in (B). Values in the graph represent means ± SEM. Each mean was determined from independent ITC binding experiments as shown in Table 1; N in Table 1 refers to the number of independent experiments for each condition.
Structure  , DOI: ( /j.str )
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5. Figure 4
The Cyclic Purine Nucleotides cPUMP and 2-NH2-cPUMP but Not cIMP Bind to the HCN2 C-Linker/CNBD with Negative Cooperativity
(A) Chemical structure of the three ligands. The atoms in the purine ring are labeled as a reference.
(B) Plots of heat and binding isotherms produced upon progressive injections of cPuMP, 2-NH2-cPuMP, and cIMP to 200 μM HCN2 C-linker/CNBD measured by ITC.
(C) Bar graph showing the affinity and thermodynamics of binding, which were determined from the fit in (B). Values represent means ± SEM. Each mean was determined from independent ITC binding experiments as shown in Table 1; N in Table 1 refers to the number of independent experiments for each condition.
Structure  , DOI: ( /j.str )
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6. Figure 5
Cyclic AMP and Analogs Variably Shift the HCN2 Ih Activation Curve to Less Negative Potentials
Plots of degree of current activation, determined by normalizing tail-current amplitudes to their maximum value (I/Imax), versus test voltages. Values represent means ± SEM and the number of cells for each condition (n values) are shown in Tables 1 and 2. The solid lines through the values represent fitting to a Boltzmann curve by Equation 1 (methods), which yielded values for V1/2 and k (Tables 1 and 2). The shift in V1/2 is smaller for cCMP and cIMP.
Structure  , DOI: ( /j.str )
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7. Figure 6
Crystal Structures of Five Analogs Bound to the HCN2 C-Linker/CNBD Reveal their Ligand-Binding Configuration and Important Interactions
The binding behavior of the five analogs to HCN2 C-linker/CNBD is visualized using X-ray crystallography with PyMOL. The PyMOL structures consist of the protein represented with a ribbon backbone and the ligand represented as sticks; residues that interact with the ligand are also represented as sticks. The blue electron density is generated by the 2mFo − DFc map of the structure, sigma cutoff at 1.0, showing how each ligand fits. Refer to Figure S2 for the 2mFo − DFc omit map. The interactions inside the five binding pockets are also presented in a simplified 2D schematics, and the pockets of cAMP (PDB: 1Q5O) and cGMP (PDB: 1Q3E) are added as a reference. Dashed lines are hydrogen bonds determined by PISA; double-headed arrows are salt bridges, and curved lines are hydrophobic interactions. The numbers categorize where the bonds are found and correspond to Table S1, which contains intermolecular distances.
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8. Figure 7
Effect of cUMP on the Binding and Oligomerization R635A Mutation in HCN2 C-linker/CNBD
(A) Plots of heat produced upon progressive injections of cAMP and cUMP to 200 μM of R635A HCN2 C-linker/CNBD measured by ITC. The inflections in the top plot arise from injections of cAMP where each inverted peak shows the heat difference between the sample and the reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plot shows values determined by integration of the area under the peaks from the upper plot versus the ratio of injected ligand to protein. The solid line through the values represents either a single-binding-site model (cUMP) or a two-independent-binding-site model (cAMP), which yielded values for affinity and energetics (ΔG, ΔH, and ΔS).
(B) Bar graph showing the thermodynamics of binding, which were determined from the fit in (A). Values in the graph represent means ± SEM. Each mean was determined from independent ITC binding experiments as shown in Table 1; N in Table 1 refers to the number of independent experiments for each condition.
(C) Shown are plots of estimated average molecular weight versus concentration of purified HCN2 C-linker/CNBD protein. The estimated molecular weight was determined by DLS and is proportional to the population of oligomers in solution. An increase in oligomerization occurs with increasing protein concentration and further increase in the presence of cAMP and cUMP. Values represent means ± SEM, determined from three separate preparations of purified protein measured on different days.
(D) Activation curves determined by plotting tail-current amplitudes, which were normalized to their maximum value (I/Imax), versus test voltage. The solid lines through the values represent fitting of a Boltzmann curve by Equation 1 (methods). Fits yielded values for V1/2 and k, which are found in Table 1 along with other parameters. Both cAMP and cUMP produce a shift in the activation curve that is comparable with the shift they produce in the wild-type channel. Values represent means ± SEM and the number of cells for each condition (n values) are shown in Tables 1 and 2.
Structure  , DOI: ( /j.str )
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