Structure and dynamics underlying elementary ligand binding events in human pacemaking channels

Abstract

Although molecular recognition is crucial for cellular signaling, mechanistic studies have relied primarily on ensemble measures that average over and thereby obscure underlying steps. Single-molecule observations that resolve these steps are lacking due to diffraction-limited resolution of single fluorophores at relevant concentrations. Here, we combined zero-mode waveguides with fluorescence resonance energy transfer (FRET) to directly observe binding at individual cyclic nucleotide-binding domains (CNBDs) from human pacemaker ion channels critical for heart and brain function. Our observations resolve the dynamics of multiple distinct steps underlying cyclic nucleotide regulation: a slow initial binding step that must select a 'receptive' conformation followed by a ligand-induced isomerization of the CNBD. X-ray structure of the apo CNBD and atomistic simulations reveal that the isomerization involves both local and global transitions. Our approach reveals fundamental mechanisms underpinning ligand regulation of pacemaker channels, and is generally applicable to weak-binding interactions governing a broad spectrum of signaling processes.DOI: http://dx.doi.org/10.7554/eLife.20797.001

Observing Single‐Molecule Dynamics at Millimolar Concentrations

“…4b). This model is consistent with X-ray crystal structures in which the C-helix caps the bound ligand [24–25] , and electron paramagnetic resonance studies that suggest similar capping of the binding site also occurs in the unliganded CNBD, thereby temporarily blocking access by fcGMP. [23] Furthermore, this scheme is similar to that observed for the binding dynamics of the higher affinity ligand fcAMP.…”

“…[23] Furthermore, this scheme is similar to that observed for the binding dynamics of the higher affinity ligand fcAMP. [24] In comparison to fcAMP at monomeric CNBDs, our high-concentration single-molecule studies reveal that the lower apparent affinity of fcGMP as compared to fcAMP is due not only to slower binding, but also to a reduction in the probability that bound ligand will induce a stabilizing isomerization of the CNBD that prolongs the total lifetime of the bound state.…”

“…As expected for a binding reaction, unbound lifetimes decreased with increasing fcGMP concentration, whereas bound lifetimes were relatively concentration independent. Based on previous observations that the CNBD isomerizes between two con-formations, [24] we compared the likelihood of several kinetic models (Fig. S2e).…”

Structures of the Human HCN1 Hyperpolarization-Activated Channel

“…Local superposition of the ligand-free and cAMP-bound CNBDs (using the β-jelly rolls) shows the conformational changes near the binding pocket that are induced by cAMP (Figure 6C). Specifically, binding of cAMP stabilizes the P-helix and causes the A-, B-, and C-helices to approach the β-jelly roll, as has been suggested based on both spectroscopic and crystallographic studies of isolated domains (Goldschen-Ohm et al, 2016; Puljung et al, 2014; Saponaro et al, 2014). In addition, helices D and E, not observed in the ligand-free cryo-EM structure, become ordered in the cAMP-bound state (compare Figures 7A and 1C).…”

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

“…Cyclic nucleotide-gated (CNG) ion channels are tetrameric potassium channels that are of particular interest due to their function in olfactory and visual signaling cascades (Kaupp and Seifert, 2002; Craven and Zagotta, 2006). However, there is only very limited data available on purified CNG channels under defined conditions, mostly from single molecule force spectroscopy (Higgins et al , 2002; Maity et al , 2015; Goldschen-Ohm et al , 2016; Mazzolini et al , 2018). Electrophysiological characterization of CNG channels expressed in Xenopus oocytes or mammalian cells (Biel et al , 1993 and 1994; Baumann et al , 1994; Weyand et al , 1994; Yu et al , 1996; Zagotta and Siegelbaum, 1996), shows that they are activated by micromolar concentrations of cyclic nucleotides (cNMP), but cAMP and cGMP can act differentially on different channel subtypes.…”