James and Zagotta discuss how recent cryoEM structures inform our understanding of cyclic nucleotide–binding domain channels.

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.pulsed EPR | spectroscopy | cryo-EM U trophin is a homolog protein of dystrophin that has shown high therapeutic promise for the treatment of muscular dystrophy (1). It is endogenously found in fetal or regenerating muscle but is replaced by dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, as the muscle matures (2). Up-regulation of utrophin in mdx mice, which lack dystrophin, has been shown to rescue its dystrophic phenotype, improving muscle morphology and function (1, 3). The full-length protein is not required to improve dystrophic pathology in mdx mice; i.e., substantial internal truncations in utrophin can be tolerated (4). These internally truncated constructs for muscular dystrophy therapeutics support the importance of actin binding by the N-terminal portions of either dystrophin or utrophin (5). Utrophin (395 kD) and dystrophin (427 kD) both contain highly homologous N-terminal actin-binding domains (ABD1), consisting of a pair of calponin homology (CH) domains. Despite additional actin-binding regions identified in the central spectrin-type repeats (6), microutrophin constructs with high potential for clinical applications rely almost exclusively on the N-terminal CH domains for actin interaction (7,8). Therefore, understanding the structural interaction between utrophin CH domains and actin has become crucial for the rational development of therapeutic constructs.More generally, there is an urgent need for a structural blueprint of CH domain-actin complexes for the entire spectrin superfamily of actin-binding proteins (e.g., fimbrin and α-actinin), of which dystrophin and utrophin are members. The diversity of crystal structur...

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxylterminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.yclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are cationpermeable ion channels regulated by the direct binding of cyclic nucleotides (cAMP or cGMP) (1). CNG channels are present in retinal photoreceptors and olfactory sensory neurons, where they perform chemoelectrical energy conversion in response to light or odor stimuli, respectively. Mutations in CNG channels have been associated with numerous inherited retinal degenerative disorders, achromatopsia, and anosmia (2). HCN channels are found in the cardiac sinoatrial node and throughout the nervous system, where they open in response to membrane hyperpolarization and generate a depolarizing current responsible for rhythmic firing (1). HCN channel mutations and mistrafficking have been associated with several disorders, including sinus bradycardia, epilepsy, and autism (3, 4).CNG and HCN channels possess a cyclic nucleotide-binding domain (CNBD) in their carboxyl-terminal region, and binding of cyclic nucleotide produces a large increase in the open probability of the channel pore. Cyclic nucleotide binding to HCN channels also shifts the voltage dependence of activation to more depolarized potentials, increasing the rate and extent of channel opening (5). CNG and HCN channels are part of a family that includes KCNH potassium channels (Fig. 1A and Fig. S1). KCNH channels, however, are distinct in that they possess a cyclic nucleotide-binding homology domain (CNBHD) occupied by an "intrinsic ligand" and are not directly regulated by cyclic nucleotides (6-8).CNG and HCN channels also harbor a C-linker domain situated between the pore and the CNBD. Based on its position, along with mutagenesis and cross-linking studies, this domain is thought t...

KCNH potassium channels possess an intrinsic ligand in their cyclic nucleotide-binding homology domain, located at the N- and C-terminal domain interface. Dai et al. show that this intrinsic ligand regulates voltage-dependent potentiation via a rearrangement between the ligand and its binding site.

We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by EPR, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state, and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here we sought to control the T/R equilibrium by an alternative means – varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB’s cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by EPR in the absence of SERCA, but also by time-resolved fluorescence resonance energy transfer (TR-FRET) from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain, an inhibitory T state that interacts strongly with the membrane surface, and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.