Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Reduced Activity of Geranylgeranyl Diphosphate Synthase Mutant Is Involved in Bisphosphonate-Induced Atypical Fractures.

Bisphosphonates are widely used for treating osteoporosis, a common disorder in which bone strength is reduced, increasing the risk for fractures. Rarely, bisphosphonates can paradoxically lead to atypical fractures occurring spontaneously or with trivial trauma. Recently, a novel missense mutation (D188Y) in the GGPS1 gene, encoding for geranylgeranyl diphosphate synthase (GGPPS), was associated with bisphosphonate-induced atypical fractures. However, the molecular basis for GGPPS involvement in this devastating condition remains elusive. Here, we show that while maintaining an overall unperturbed global enzyme structure, the D188Y mutation leads to ∼4-fold catalytic activity decrease. Furthermore, GGPPS-D188Y is unable to support cross-species complementation, highlighting the functional significance of the reduced catalytic activity observed in vitro. We next determined the crystal structure of apo-GGPPS-D188Y, revealing that while Y188 does not alter the protein fold, its bulky side chain sterically interferes with substrate binding. In agreement, we show that GGPPS-D188Y exhibits ∼3-fold reduction in the binding affinity of zoledronate, a commonly used bisphosphonate. However, inhibition of the mutated enzyme by zoledronate, in pharmacologically relevant concentrations, is maintained. Finally, we determined the crystal structure of zoledronate-bound GGPPS-D188Y, revealing large ligand-induced binding pocket rearrangements, revising the previous model for GGPPS-bisphosphonate interactions. In conclusion, we propose that among heterozygotes residual GGPPS activity is sufficient to support physiologic cellular function, concealing any pathologic phenotype. However, under bisphosphonate treatment, GGPPS activity is reduced below a crucial threshold for osteoclast function, leading to impaired bone remodeling and increased susceptibility to atypical fractures.

Ultrasensitive chemiluminescent neuraminidase probe for rapid screening and identification of small-molecules with antiviral activity against influenza A virus in mammalian cells.

Influenza A virus is the most virulent influenza subtype and is associated with large-scale global pandemics characterized by high levels of morbidity and mortality. Developing simple and sensitive molecular methods for detecting influenza viruses is critical. Neuraminidase, an exo-glycosidase displayed on the surface of influenza virions, is responsible for the release of the virions and their spread in the infected host. Here, we present a new phenoxy-dioxetane chemiluminescent probe (CLNA) that can directly detect neuraminidase activity. The probe exhibits an effective turn-on response upon reaction with neuraminidase and produces a strong emission signal at 515 nm with an extremely high signal-to-noise ratio. Comparison measurements of our new probe with previously reported analogous neuraminidase optical probes showed superior detection capability in terms of response time and sensitivity. Thus, as far as we know, our probe is the most sensitive neuraminidase probe known to date. The chemiluminescence turn-on response produced by our neuraminidase probe enables rapid screening for small molecules that inhibit viral replication through different mechanisms as validated directly in influenza A-infected mammalian cells using the known inhibitors oseltamivir and amantadine. We expect that our new chemiluminescent neuraminidase probe will prove useful for various applications requiring neuraminidase detection including drug discovery assays against various influenza virus strains in mammalian cells.

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge.

Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]-related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the ß-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge's crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface.